In Bolivia, there are currently three types of Creole cattle: Altiplano, Valleys, and Plains. The cattle of the Valleys comprise isolated populations which have been little studied so far. The objective of the present study was to perform the genetic characterization of the Creole cattle from the Valleys of Santa Cruz. We analyzed 17 autosomal microsatellites (STRs), 5 STRs and one indel of the Y chromosome and a fragment of the D-Loop region of mitochondrial DNA. DNA was extracted from 98 Creole animals belonging to: 25 from the population of the Valleys of Santa Cruz, 35 from Yacumeño Creole, 17 from Saavedreño Creole, and 21 from Creole of the Centro de Ecología Aplicada Simón I. Patiño (CEASIP). The 17 autosomal loci studied were polymorphic. The average number of alleles per locus was 5.18, ranging from two to 10. The Heterozygosis values observed in the studied STRs varied between 0.083 and 0.898, resulting in an average heterozygosity of 0.664. The analysis of the Y chromosome markers allowed the detection of two haplotypes, one from B.taurus origin (Y2 haplogroup; - Val1) and other of zebu origin (Y3 haplogroup). The analysis of the maternal lineages detected ten mitochondrial haplotypes, three belonging to the European haplogroup T3 and seven to the African haplogroup T1. The analysis of genetic distances and the principal component analysis, based on microsatellite data, showed that the population studied exhibited a larger divergence with respect to the other Bolivian Creole cattle populations. Maternal genetic composition showed a mixed European and African origin. Finally, the analysis of the Y chromosome, as well as the Structure analysis showed that the population of Creole cattle of the Valley showed introgression of Zebu genes. The study can be the starting point to create conservation programs for the Creole cattle from the valleys of Santa Cruz, which is an important animal resource that must be conserved.