One hundred seventy-eight mycoplasma strains isolated from South African poultry flocks between 2003 and 2015 were identified by full-genome sequencing and phylogenetic analysis of the 16S rRNA gene and were classified as follows: (25%), (25%), , (23%), (14%), (10%), and (3%), as well as one strain (1%). MIC testing was performed on the axenic samples, and numerous strains of each species were resistant to either chlortetracycline or tylosin or both, with variable sensitivity to enrofloxacin. The strains of all species tested remained sensitive to tiamulin, except for one sample that demonstrated intermediate sensitivity. The mutation of A to G at position 2059 (A2059G) in the 23S rRNA gene, which is associated with macrolide resistance, was found in the South African and strains, as well as a clear correlation between macrolide resistance in and and mutations G354A and G748A in the L4 ribosomal protein and 23S rRNA gene, respectively. No correlation between resistance and point mutations in the genes studied could be found for Only a few strains were resistant to enrofloxacin, apart from one strain with point mutation D420N, which has been associated with quinolone resistance, and no other known markers for quinolone resistance were found in this study. Proportionally more antimicrobial-resistant strains were detected in ,, and than in and Of concern, three strains showed multidrug resistance to chlortetracycline, tylosin, and oxytetracycline. Nonpathogenic poultry species are often overlooked due to their lesser impact on poultry health and production compared to the OIE-listed pathogenic strains and The use of antimicrobials as in-feed growth promoters and for the control of mycoplasmosis is common in poultry production across the world. Here, we provide evidence that certain nonpathogenic species are acquiring multidrug resistance traits. This would have significant implications if these species, for which no vaccines are applied, are able to transfer their antibiotic resistance genes to other mycoplasmas and bacteria that may enter the human food chain.