Mycoplasma spp. can cause diseases of the respiratory system as well as urogenital infections, infertility, and anemia. The members of this genus have a low G + C content compared to other bacteria. Because primers used in the random amplified polymorphic DNA (RAPD) technique are only 10 bp long and have high GC content, this method can be inadequate for genotyping Mycoplasma spp. isolates. The aim of this study was to develop and evaluate multiple-locus variable number tandem repeat analysis (MLVA) and two-primer RAPD (TP-RAPD) procedures for subtyping Mycoplasma cynos isolates. A total of 55 M. cynos isolates obtained from 162 bronchoalveolar lavage fluid samples from shelter and pet dogs were used in this study. Seventy-four tandem repeat regions were detected in the M. cynos genome, and two of these loci were determined to be suitable and used for development of the MLVA scheme. The results of variable number tandem repeat (VNTR) analysis and TP-RAPD-PCR were compared with RAPD-PCR. The discriminatory power of TP-RAPD-PCR (Hunter-Gaston diversity index [HGDI] = 0.84) was higher than those of RAPD-PCR (HGDI = 0.727), VNTR1 (HGDI = 0.8), and VNTR3 (HGDI = 0.757). We observed that the TP-RAPD-PCR and MLVA methods provide clearer data and are more successful in determining genetic diversity, in contrast to the RAPD-PCR method for this species.