In order to investigate the infection and genetic variation of Mycoplasma bovis in central and southern Ningxia，495 bovine nose swabs collected from intensive farms，slaughterhouses and trading markets in nine counties/districts in the region were detected by PCR and identified through strain isolation. The results showed that 59 positive samples were detected，with an overall positive rate of 11.92%，the positive rate of all the counties/districts was 1.82% to 21.82%，specifically，11.11%，13.33% and 12.59% for intensive farms，slaughterhouses and trading markets，respectively；16 suspected strains of Mycoplasma bovis were isolated，then amplified by oppF gene-specific primers，sequenced and analysed to establish a phylogenetic tree，4 strains were identified as Mycoplasma bovis；the phylogenetic tree was further established by 16S rRNA sequence of the isolated strain and the representative strain of Mycoplasma bovis，and it was observed that the strain was most closely related to the international standard strain PG45 and Ningxia strain（Ningxia-1）. After drug sensitivity test，it was found that the isolated strain was not sensitive to oxacillin，streptomycin，ciprofloxacin，polymyxin B，amoxicillin，piperacillin，cefazolin and ampicillin. In conclusion，Mycoplasma bovis was widely distributed in central and southern Ningxia，and no major genetic variation was observed in the prevalent strains that were resistant to some antibiotics. It was thus recommended to strengthen the prevention and control for bovine mycoplasma disease，and select sensitive drugs with joint and interspersed administration in combination with drug sensitivity test during clinical prevention to avoid any drug resistance. A reference was therefore provided for future prevention and control for bovine mycoplasma disease and clinical administration in the region.