Background/Objectives: Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major human pathogen. This study aimed to determine the phenotypic profiles of macrolide resistance in S. pyogenes isolates from southeastern Turkey and to investigate underlying genotypic mechanisms using Oxford Nanopore sequencing. Methods: A total of 83 clinical isolates of S. pyogenes were collected between January 2024 and June 2025. Antimicrobial susceptibility was assessed by disk diffusion following EUCAST guidelines. Macrolide resistance phenotypes (iMLSB, cMLSB, M) were determined by the D‑zone test. Whole‑genome sequencing of erythromycin‑resistant isolates was performed using Oxford Nanopore technology, and resistance determinants were analyzed with the CARD/RGI pipeline.Results: All isolates were susceptible to penicillin. Six isolates (7.2%) exhibited erythromycin resistance: three iMLSB, two cMLSB, and one M phenotype. Adult isolates were exclusively iMLSB, whereas pediatric isolates included one M and two cMLSB phenotypes. Genotypic analysis revealed erm(A) in all iMLSB isolates, mef(A)/msr(D) in the M phenotype, and absence of major resistance genes in cMLSB isolates, which instead carried chromosomal lmrP and mef(E)‑like sequences. Co‑occurrence of tetracycline resistance genes (tetM/O) was observed in several isolates.Conclusions: Penicillin remains the cornerstone of GAS therapy; however, macrolide resistance persists through diverse mechanisms. The coexistence of erm(A)‑mediated iMLSB, mef(A)/msr(D)‑associated M phenotype, and unexplained cMLSB resistance highlights the need for routine D‑testing and genomic surveillance. These findings emphasize the importance of integrating phenotypic and genotypic approaches to monitor resistance evolution in GAS.