Next-generation sequencing (NGS) has revolutionized molecular ecology by enabling the rapid and cost-effective generation of large-scale sequencing data from non-model organisms. This review discusses the applications of NGS in ecological, population genetic, and conservation genetic studies of species with limited genomic resources. NGS can be used for genomic or transcriptomic approaches, allowing the study of nucleotide variation, transcriptome characteristics, and gene expression levels. Transcriptome characterization is the most common application in non-model species, involving the identification of expressed genes and their functional roles. NGS enables the discovery of genetic markers and facilitates the study of quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS). It also improves the analysis of population history, genetic structure, and relatedness. NGS data can be used for genome-wide scans, comparative genomics, and the study of gene regulation, DNA-protein interactions, and epigenetics. The availability of genomic resources for related species enhances the utility of NGS in non-model species. NGS has been applied to various organisms, including wasps, butterflies, plants, and fish, to study ecological and evolutionary questions. Transcriptome sequencing has been used to identify candidate genes for traits such as food provisioning and foraging behavior, as well as to study immune responses and pathogen resistance. NGS also enables the development of molecular markers, such as SNPs and microsatellites, for population genetic studies. The use of NGS in targeted sequencing allows for the study of specific loci, improving the efficiency of population genetic analyses. NGS has also been used for whole genome sequencing (WGS) in non-model species, although this is more challenging due to the lack of genomic resources. The use of NGS in molecular ecology is expanding, with new technologies and approaches enabling the study of a wide range of ecological and evolutionary questions. However, challenges remain, including the need for careful planning, data analysis, and the selection of appropriate sequencing platforms. Overall, NGS is a powerful tool for molecular ecology, enabling the study of non-model organisms and providing insights into ecological and evolutionary processes.Next-generation sequencing (NGS) has revolutionized molecular ecology by enabling the rapid and cost-effective generation of large-scale sequencing data from non-model organisms. This review discusses the applications of NGS in ecological, population genetic, and conservation genetic studies of species with limited genomic resources. NGS can be used for genomic or transcriptomic approaches, allowing the study of nucleotide variation, transcriptome characteristics, and gene expression levels. Transcriptome characterization is the most common application in non-model species, involving the identification of expressed genes and their functional roles. NGS enables the discovery of genetic markers and facilitates the study of quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS). It also improves the analysis of population history, genetic structure, and relatedness. NGS data can be used for genome-wide scans, comparative genomics, and the study of gene regulation, DNA-protein interactions, and epigenetics. The availability of genomic resources for related species enhances the utility of NGS in non-model species. NGS has been applied to various organisms, including wasps, butterflies, plants, and fish, to study ecological and evolutionary questions. Transcriptome sequencing has been used to identify candidate genes for traits such as food provisioning and foraging behavior, as well as to study immune responses and pathogen resistance. NGS also enables the development of molecular markers, such as SNPs and microsatellites, for population genetic studies. The use of NGS in targeted sequencing allows for the study of specific loci, improving the efficiency of population genetic analyses. NGS has also been used for whole genome sequencing (WGS) in non-model species, although this is more challenging due to the lack of genomic resources. The use of NGS in molecular ecology is expanding, with new technologies and approaches enabling the study of a wide range of ecological and evolutionary questions. However, challenges remain, including the need for careful planning, data analysis, and the selection of appropriate sequencing platforms. Overall, NGS is a powerful tool for molecular ecology, enabling the study of non-model organisms and providing insights into ecological and evolutionary processes.