The objective of this study was to determine the genetic diversity of creole cattle in northwestern Mexico using the BoLA-DRB3.2 locus of the Major Histocompatibility Complex (MHC). A total of 56 creole cattle were sampled from five communities; in the state of Chihuahua (Cerocahui, Guadalupe y Calvo and Cuauhtémoc) and in the state of Baja California Sur (La Paz and Mulegé). The BoLA-DRB3.2 locus was genotyped by PCR-RFLP assay. Thirty-nine alleles were identified, out of which 14 had not been previously reported. The average level of inbreeding in all populations analyzed was F
IS = 0.09 (P < 0.0001), but only two populations (Cerocahui and Guadalupe y Calvo) showed an excess of homozygotes (P < 0.05). The breed differentiation in all populations studied was F
SC = 0.068 (P < 0.0001). The smallest genetic distance was between La Paz and Mulegé (0.022); but Mulegé presented smaller distances (0.028–0.053) with the populations of La Paz (0.071–0.083) and with Chihuahua. Baja California Sur populations are grouped in a separate branch than Chihuahua populations. We conclude that creole cattle from Baja California Sur and Chihuahua show high genetic diversity in the locus BoLA-DRB3.2.