Physalis peruviana L. often known as goldenberry, has increased its commercial growth in the international market in recent years due to its nutritional value and antioxidant potential. This situation has enabled countries such as Peru to increase their production in order to meet the global demand. However, investigations about the genetic diversity of cultivated and wild populations of goldenberry are still in their early stages. FISH mapping of 5s and 45s rDNA loci and flow cytometry estimation of nuclear DNA content were used to assess genetic differences between wild and cultivated goldenberry populations from Ayacucho and Cajamarca. The majority of metaphases had six 5s rDNA sites for all populations and two and four 45s rDNA sites for the cultivated and wild populations, respectively. We were able to characterize nine different types of chromosomes based on their morphology, fluorescence, rDNA location, and conservation across populations by analyzing the chromosomes that contained rDNA. Furthermore, cultivated populations had more nuclear DNA (13.262±0.087 pg) than wild populations (12.955±0.086 pg). The results show genetic differences between wild and cultivated populations of goldenberry at molecular cytogenetic level as well as in genome size. These findings establish a precedent for future cytogenetic and genomic studies in goldenberry populations, enabling future breeding programs.