This study describes the development of PCR primers to detect erythromycin resistance determinants in clinical isolates. Erythromycin resistance can arise through three mechanisms: target modification by rRNA methylases, inactivating enzymes like erythromycin esterases (ereA, ereB) and macrolide phosphotransferases (mphA), and efflux pumps such as msrA/msrB and mefA/mefE. The study designed specific primers to distinguish between these mechanisms, enabling direct sequencing of amplified PCR products for comparison of resistance determinants in clinical strains. The methodology allows for the simultaneous detection of multiple resistance determinants in a single PCR reaction, facilitating surveillance of erythromycin resistance. The primers were designed to detect macrolide efflux (msrA/msrB or mefA/mefE) and drug inactivation (ereA, ereB, mphA) as well as to distinguish between ermA, ermB, and ermC classes in pathogenic bacteria. The study tested these primers on reference strains and clinical isolates of various bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae. The results showed that the primers could effectively detect the presence of specific resistance determinants, with clear PCR product sizes distinguishing between different mechanisms. The study also demonstrated that the PCR methodology could be used to identify resistance determinants in clinical isolates, providing a powerful tool for surveillance and understanding of erythromycin resistance. The results support the use of PCR in clinical microbiology for the detection of resistance mechanisms, which can aid in the appropriate prescription of antibiotics and reduce the development of resistance. The methodology is also useful for direct sequencing of amplified PCR products, allowing for the comparison of resistance determinants across different bacterial species.This study describes the development of PCR primers to detect erythromycin resistance determinants in clinical isolates. Erythromycin resistance can arise through three mechanisms: target modification by rRNA methylases, inactivating enzymes like erythromycin esterases (ereA, ereB) and macrolide phosphotransferases (mphA), and efflux pumps such as msrA/msrB and mefA/mefE. The study designed specific primers to distinguish between these mechanisms, enabling direct sequencing of amplified PCR products for comparison of resistance determinants in clinical strains. The methodology allows for the simultaneous detection of multiple resistance determinants in a single PCR reaction, facilitating surveillance of erythromycin resistance. The primers were designed to detect macrolide efflux (msrA/msrB or mefA/mefE) and drug inactivation (ereA, ereB, mphA) as well as to distinguish between ermA, ermB, and ermC classes in pathogenic bacteria. The study tested these primers on reference strains and clinical isolates of various bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae. The results showed that the primers could effectively detect the presence of specific resistance determinants, with clear PCR product sizes distinguishing between different mechanisms. The study also demonstrated that the PCR methodology could be used to identify resistance determinants in clinical isolates, providing a powerful tool for surveillance and understanding of erythromycin resistance. The results support the use of PCR in clinical microbiology for the detection of resistance mechanisms, which can aid in the appropriate prescription of antibiotics and reduce the development of resistance. The methodology is also useful for direct sequencing of amplified PCR products, allowing for the comparison of resistance determinants across different bacterial species.