Mycoplasmas cause the most severe and economically costly diseases of cattle throughout the world. In this study, Mycoplasma species were isolated from calves and cows with suspected mycoplasmosis in Holstein dairy cattle farms within 7 geographical regions of Turkey between May 2010 and December 2015. Mycoplasma infections were positive in 17 (80.9%) of 21 dairy cattle farms and the overall percentage was calculated as 32.1%. The highest isolation rate occurred in the Southeastern Anatolia Region (42.8%), and the lowest was observed in the Mediterranean Region (19.6%). In total, 172 Mycoplasma spp. were isolated from samples. Using PCR analysis, 149 (87.6%) isolates were identified as Mycoplasma bovis (M. bovis). Eleven (6.3%) isolates were identified as M. alkalescens, 2 (1.1%) were M. canis, and 10 (5.8%) were M. bovigenitalium. The isolation rate was found to be increasing annually. In conclusion, mycoplasmosis is a common problem in Holstein dairy cattle farms in Turkey, and M. bovis is the most frequently encountered cause of mycoplasma infections. The isolation rate seems to have increased in correlation with increased live cattle imports. Additionally, M. alkalescens and M. canis were isolated and identified in respiratory tract infections in cattle from Turkey for the first time.