The polymorphism of locus BoLA-DRB3.2 of the Major Histocompatibility Complex was evaluated in two northern Mexican Creole cattle populations, Chihuahua (n = 47) and Tamaulipas (n = 51). The BoLA-DRB3.2 locus was typed by amplification and digestion with restriction endonuclease enzymes (PCR-RFLP). Fifty-two alleles were detected (28 previously reported and 24 new ones). In the Chihuahua population, 18 alleles and 5.5 effective alleles were found, while in the Tamaulipas population there were 34 and 10.8, respectively. The allele frequencies ranged from 0.011 to 0.383 in Chihuahua and from 0.010 to 0.206 in Tamaulipas. The frequencies of the new alleles in both cattle populations were low (0.010 to 0.053). The expected heterozygosity was 0.827 and 0.916, respectively, for the Chihuahua and Tamaulipas populations. Both populations presented a heterozygote deficit: [Chihuahua F IS = 0.1 (p = 0.019) and Tamaulipas F IS = 0.317 (p < 0.001)]. In conclusion, this study showed that the Mexican Creole cattle have many low-frequency alleles, several of which are exclusive to these populations. Genetic distances obtained show that the Mexican Creole cattle population is composed of independent populations, far apart from other South American Creole populations.