The complete mitochondrial D-loop sequences, 910 bp in length, in 82 individual cattle from 4 breeds in Guizhou province were analyzed. The results revealed 31 mitochondrial haplotypes, 65 polymorphic sites, covering 7.14% of the entire length of the sequence. Among these polymorphic sites, there were 62 transitions, 2 transversions and 1 coexistent site of transition and transversion. The nucleotide diversity (pi value) and haplotype diversity (H) estimated from mtDNA D-loop region in 4 cattle breeds in Guizhou varied from 2.16%-2.61% and 0.695-0.909, respectively, showing that abundant mitochondrial genetic diversity exists in Guizhou cattle breeds. The Neighbor-Joining molecular phylogenetic tree of mtDNA D-loop of 4 Guizhou cattle breeds was constructed according to the 31 haplotypes. The NJ tree indicated that the origin of cattle breeds was from Bos taurus and Bos indicus which had nearly the same influence on cattle breeds in Guizhou. The feasibility of applying nucleotide diversity (pi value) to the evaluation of bovine genetic differentiation was discussed.