Wolbachia are intracellular bacteria that infect a wide range of arthropods and are associated with various reproductive phenotypes, including cytoplasmic incompatibility, parthenogenesis, and feminization. While it is known that these bacteria form a single clade within the α-Proteobacteria, the evolutionary relationships between different Wolbachia strains have not been fully resolved. This study reports the cloning and sequencing of the wsp gene, which encodes a surface protein of Wolbachia, from 28 representative strains. The wsp gene sequences showed high variability and were used to resolve the phylogenetic relationships of different Wolbachia strains. Based on these sequences, the Wolbachia pipientis clade is divided into 12 groups. The study also presents a method for classifying Wolbachia using group-specific wsp PCR primers, which allows for the typing of Wolbachia isolates without cloning and sequencing individual genes. This system facilitates future studies on the distribution and biology of Wolbachia strains. The wsp gene is a fast-evolving gene that provides more informative characters for phylogenetic analysis compared to previously used genes like 16S rRNA and flsZ. The study also discusses the potential for selection pressure on the wsp gene and its implications for phylogenetic analysis. The wsp gene sequences were used to identify distinct Wolbachia clades within the A and B groups. The study proposes a naming system for Wolbachia groups based on wsp sequence similarity. The results suggest that the wsp gene is a valuable tool for Wolbachia classification and phylogenetic analysis. The study also highlights the potential of wsp sequences for predicting other phenotypes such as parthenogenesis. The use of wsp sequences as diagnostic and evolutionary tools has significant implications for understanding the biology of Wolbachia. The study also discusses the importance of expanding the number of sequenced Wolbachia variants to define a comprehensive system of group-specific primer sets. The study was supported by grants from various funding sources.Wolbachia are intracellular bacteria that infect a wide range of arthropods and are associated with various reproductive phenotypes, including cytoplasmic incompatibility, parthenogenesis, and feminization. While it is known that these bacteria form a single clade within the α-Proteobacteria, the evolutionary relationships between different Wolbachia strains have not been fully resolved. This study reports the cloning and sequencing of the wsp gene, which encodes a surface protein of Wolbachia, from 28 representative strains. The wsp gene sequences showed high variability and were used to resolve the phylogenetic relationships of different Wolbachia strains. Based on these sequences, the Wolbachia pipientis clade is divided into 12 groups. The study also presents a method for classifying Wolbachia using group-specific wsp PCR primers, which allows for the typing of Wolbachia isolates without cloning and sequencing individual genes. This system facilitates future studies on the distribution and biology of Wolbachia strains. The wsp gene is a fast-evolving gene that provides more informative characters for phylogenetic analysis compared to previously used genes like 16S rRNA and flsZ. The study also discusses the potential for selection pressure on the wsp gene and its implications for phylogenetic analysis. The wsp gene sequences were used to identify distinct Wolbachia clades within the A and B groups. The study proposes a naming system for Wolbachia groups based on wsp sequence similarity. The results suggest that the wsp gene is a valuable tool for Wolbachia classification and phylogenetic analysis. The study also highlights the potential of wsp sequences for predicting other phenotypes such as parthenogenesis. The use of wsp sequences as diagnostic and evolutionary tools has significant implications for understanding the biology of Wolbachia. The study also discusses the importance of expanding the number of sequenced Wolbachia variants to define a comprehensive system of group-specific primer sets. The study was supported by grants from various funding sources.