'Ancho' type chilis (Capsicum annuum L. var. annuum) are an important ingredient in the traditional cuisine of Mexico and so are in high demand. It includes six native sub-types with morphological and fruit color differences. However, the genetic diversity of the set of these subtypes has not been determined. The objective of this study was to characterize the genetic diversity of native Mexican ancho chili populations using microsatellites and to determine the relationship among these populations. Twenty-four microsatellite loci were used to analyze 38 native populations of 'Ancho' chilis collected in seven states of Mexico; three populations different from the ancho type ('Piquin', 'Guajillo', and 'Chilaca') and three hybrids (Capulin, Abedul, and green pepper) were included as controls. The number of alleles per locus, number and percentage of polymorphic loci, polymorphic information content (PIC), expected heterozygosity, and Wright F statistics were obtained. Moreover, an analysis of principal components and a cluster analysis were carried out. We detected 220 alleles, with an average of 9.2 alleles per locus; PIC varied between 0.07 and 1, and expected heterozygosity was between 0.36 and 0.59. Also we identified 59 unique alleles and eight alleles common to all of the populations. The F statistics revealed broad genetic differentiation among populations. Both the analysis of principal components and the cluster analysis were able to separate the populations by origin (southern, central, and northern Mexico). The broad genetic diversity detected in the native ancho chili populations of Mexico was found in greater proportion within the populations than between populations.