In the absence to date of studies on genetic variability in the Creole cattle of the Southern Region of Ecuador, the analysis and characterization of this population was carried out in order to provide the technical information needed for the planning and development of measures aimed at the population's conservation and sustainable use in the harsh ecosystem these animals inhabit. For this purpose, 46 adult animals, out of a population of 114 Creole cattle, were genetically analysed; on the basis of phaneroptical traits, they were classified into four groups known locally as Negro Lojano (15 animals), Encerado (15), Colorado (9) and Pintado or Cajamarca (7). The analysis of the intra-population genetic variability brought out a Na of 228, an average number of alleles per locus of 8.14 and a PIC of 0.70 ± 0.10. All 28 markers analyzed were polymorphic and highly informative. The He and Ho amounted to 0.74 ± 0.0178 and 0.676 ± 0.013, which is an indication of a high molecular diversity in the population studied. The HWE determines that 32 per cent of the alleles are not in equilibrium. The Gst amounted to 4.5 ± 2.8 per cent and the Fst was 0.08 per cent, which corroborates that there is no genetic differentiation among the four groups of this population. The average values for Fit and Fis were 8.89 and 8.92 percent, respectively. The degrees of genetic distance between this Creole population and 18 cattle and zebu breeds were also determined. The distance exceeded 0.10 for all populations, with the lowest distances being those between Ec:BC (0.1031), Ec:PAJ (0.1132), Ec:NAN (0.1302), Ec:VCA (0.1326) and Ec:FRI (0.1362). The greatest distances were observed between Ec:GUZ (0.4725) and Ec:NEL (0.4624). These values show that the Creole cattle of the Southern Region of Ecuador are very distant from the zebu breeds, and therefore their ancestors would be more likely to have originated from Iberian populations. The results concerning variability and genetic distance will enable strategies to be devised for managing and conserving the Creole breed of the Southern Region of Ecuador. These results also suggest that the four groups with phaneroptical differences should be managed independently at the different altitudes of the Andean Region of Ecuador.