Polymorphisms in the dopamine β-hydroxylase gene (DBH) have been associated with temperament in cattle. In 16 animals of Colombian creole breed Blanco Orejinegro (BON), 20 Brahman (BRA) and 16 fighting bulls (GLI), polymorphisms of exon XII of the DBH gene were evaluated by sequencing. 13 haplotypes were found, 6 in BON, 9 and 2 in BRA and GLI respectively. Haplotype H2 was the most frequent in the whole population (0,615) and it was found in all breeds. Haplotypes H6 and H9 were shared between BON-BRA and BRA-GLI, respectively. The greatest value of genetic(DG: 0.915 ± 0.04) and nucleotide (DN: 0.011 ± 0.006) diversity, was found in BRA, followed by BON (DG: 0.700 ± 0.12; DN: 0.008 ± 0.004) and GLI (DG: 0.350 ± 0.14;DN: 0.0011 ± 0.001). The D-Tajima test was greater than zero, but not significant (P> 0.05) in BON and BRA, GLI presented a value of −1.92 (P < 0.05). The analysis of molecular variance showed a variation between the breeds of 23.9% and a poblacional structure (FST) of 0.23 (P < 0.001). The highest values of the coancestry coefficient were presented between GLI and BRA (0.52) and between GLI and BON (0.22). It is concluded that the GLI race has low diversity in the DBH gene compared to BON and BRA, that there are effects of neutral selection in BON and BRA breeds, whereas, in the GLI and not in favor of temperament.