Reithrodontomys spectabilis is an endemic rodent inhabiting Cozumel island in the Mexican Caribbean. This species is categorized as Threatened by Mexican law and as Critically Endangered by the IUCN. We report 10 polymorphic microsatellite loci, which were tested in 30 individuals trapped on the island. Results show a number of alleles ranging from 3 to 14, an observed heterozygosity of 0.200–0.967 and expected heterozygosities from 0.396 to 0.899. Two loci showed deviation from Hardy–Weinberg equilibrium. None of the loci showed linkage disequilibrium. These new microsatellite loci for R. spectabilis are vital to investigate different aspects of genetic diversity and help conservation of this endemic, threatened species.