Mexico has a wealth of Capsicum species, which has led to the development of a large number of chili pepper landraces. A great wealth of Capsicum germplasm can be found in southern Mexico in the Yucatan Peninsula, an important area of diversification of Capsicum annuum. Specifically, in the western Yucatan Peninsula, three of the five domesticated species of Capsicum (C. annuum, C. chinense and C. frutescens) have been reported. However, information on their genetic diversity, conservation status and potential use is lacking. To generate useful information toward the sustainable use, management and conservation of these species, we evaluated the structure, diversity and genetic relationships of nine accessions of Capsicum spp., of major importance cultivated in the western Yucatan Peninsula using 42 ISSR loci. The results indicated that these accessions consisted of three genetic groups that were defined by the respective species of each accession. The level of genetic diversity was moderate and distributed mainly among accessions. The ISSR markers detected a high level of polymorphism and allowed the genetic differentiation of the C. annuum complex. The results indicated that the accessions collected in the western Yucatan Peninsula constitute a valuable genetic resource that can be used in genetic improvement and conservation programs.