Crop wild relatives are conserved in germplasm banks as original accessions or their ex situ regenerated progenies. The commonly used sexual regeneration method in wild potatoes (FAO 2013) -cultivation of 20-25 plants/accession (N), hand-pollination with pollen mixtures when a percentage of the plants are in bloom, and composition of the regenerated accession without controlling the number of seeds provided by each female progenitor-does not consider the action of internal reproductive barriers. However, wild potatoes -mostly diploid and obligate out-crossers-may present internal pre-zygotic, post-zygotic, or both types of complete or incomplete reproductive barriers, which can reduce the effective number of progenitors (Ne) in relation to N. To evaluate possible phenotypic and genetic changes during a sexual reproduction cycle, two regeneration protocols were followed with one accession of Solanum chacoense Bitter: the current one and the proposed by Camadro (2012). The original accession and the two regenerated populations were morphologically and molecularly characterized. Twenty-one morphological characters (quantitative and qualitative) were considered, and the data were subjected to univariate and multivariate analyses. Allele frequencies were calculated for six microsatellite markers (SSR) and compared among populations with a chi-square test (α= 0.05). Great morphological variability for morphological phenotypes and four of the six analyzed SSR, as well as changes in allele frequencies (including allele loss) were revealed within and among the three populations. Therefore, it is advisable to consider aspects of reproductive and population genetics in developing regeneration protocols to prevent or reduce the risks of genetic erosion in sexual regeneration cycles.