In the Western Pyrenees, three out of four native cattle breeds are in grave danger of extinction. Genetic variation of all four breeds was assessed by analyzing 478 animals using 11 microsatellite markers. A moderate/high within-breed variability was found, a favorable factor to consider when planning conservation and improvement programs. Interestingly, the only selected commercial breed, the Pirenaica, showed depressed heterozygosity levels and a low average number of alleles, perhaps explainable by intensive human selection exacerbated by a bottleneck effect. The Pirenaica also exhibited pronounced genetic differences and was the largest contributor of diversity among the breeds from the Western Pyrenees. Among endangered cattle breeds from this region, our results highlight the singularity of the Betizu. Geographic isolation among herds may be responsible for the large F(IS) value found in the Betizu breed. Lastly, our study suggests that the use of highly selected breeds may be one of the causes of distortion in phylogenetic analyses.