In the United States, Multiple-Locus Variable number tandem repeat Analysis (MLVA) and complete P146 gene sequence are employed to characterize Mycoplasma hyopneumoniae from clinical samples. However, a comparison of MLVA and P146 sequencing and interpretation of assay results has not been conducted. Therefore, the aim of this study was to characterize and compare M. hyopneumoniae variants detected in the Midwestern United States using MLVA and P146 gene sequencing. A total of 160 samples were analyzed for this investigation. Both molecular techniques were compared for assay sensitivity, discriminatory power, and congruence. Epidemiological relationships in clustering of M. hyopneumoniae variants were evaluated employing Principal Coordinate Analysis. Fair agreement (κ = 0.34) in assay outcome was calculated between the two techniques. Ability to obtain a VNTR type or a P146 sequence was dependent upon the relative bacterial load (i.e., Ct value) in the sample. Simpson's diversity index was higher for MLVA (D = 0.899) than for P146 sequencing (D = 0.844). High congruence for the number of tandem repeats detected in the poly-serine region of P146 was also obtained. Similar epidemiological inferences were generated from the two assays, as production flow explained most of the variation in the clustering of VNTR types and P146 sequences. Results from this study highlighted differences in sensitivity and discriminatory power between the two molecular techniques. Nevertheless, both techniques revealed a wide genetic diversity among M. hyopneumoniae variants, with similar epidemiological inferences generated. Further research utilizing whole-genome sequencing could help identify other areas within the genome that are unrepresented by MLVA and P146 sequencing, can aid characterizing M. hyopneumoniae variants.