Mycoplasma gallisepticum and Mycoplasma synoviae are listed by the World Organisation for Animal Health (OIE) as notifiable avian diseases agents and are considered as economically important species affecting the South African poultry industry. A decreased number of cases identified by culture with growth inhibition of M. gallisepticum and M. synoviae and an increase in unidentified mycoplasma species were observed at the University of Pretoria diagnostic laboratory. Samples were isolated from chickens displaying typical signs associated with pathogenic mycoplasma infection, thus necessitating a closer look at the lesser known mycoplasma species found in poultry. The aim of this study was to use second generation sequencing to identify and compare mycoplasma species isolated from commercial chickens in South Africa. Mycoplasma samples were isolated, sequenced and de novo assembled for identification by 16S rRNA phylogeny. A total of 124 samples were received between 2003 and 2015, 44 of these samples contained multiple species resulting in 179 isolates identified as M. gallisepticum (24.58%), M. synoviae (9.50%), M. gallinaceum (24.02%), M. gallinarum (24.58%), M. pullorum (13.97%) and M. iners (2.79%) and one Acholeplasma laidlawii (0.56%). Antimicrobial resistance (AMR) to chlortetracycline, enrofloxacin, tylosin and tiamulin and the genes involved in AMR were studied. Three M. gallinaceum samples showed possible multidrug resistance and novel point mutations associated with AMR in M. gallinaceum and M. gallinarum were identified. The first complete genome of M. pullorum was assembled, annotated and published. Draft genome assemblies for axenic strains were constructed and candidate genes that can be tested for novel diagnostic and vaccine targets were identified.