Chile peppers are one of the most important crops in Mexico and a plethora of ecotypes can be found there. Most of them are ancient open-pollinated (OP) landraces selected by farmers for uniform phenotype but with an inherent level of genetic diversity, called criollos. In this work 15 pepper accessions, encompassing 2 criollo lines, their open-pollinated progenies, and 5 controls, were characterized with a set of 36 IPGRI descriptors and 23 SSR markers to assess the effect of open pollination in the inbreeding process. Heterozygosity levels were comprised between 12 and 47% in the progenies, which were similar or lower than those values from parent plants and similar or higher than control cultivars. Also, both progenies and parents showed similar levels of agronomic and morphological uniformity. Our results suggest that this OP program is efficient in terms of reaching enough agronomic uniformity in criollo Ancho peppers while preserving certain genetic diversity to confer adaptation to climate change.