Physalis angulata L., Physalis peruviana L., and Physalis ixocarpa Brot. ex Hornem. are cultivated Solanaceae species of nutritional, medicinal, and genetic resource importance. This study characterized germplasm accessions of these three species using chromosome counting, CMA/DAPI banding, and flow cytometry. Chromosome counts revealed 2n = 24 in the P. ixocarpa accessions IX1 and IX2, whereas 2n = 48 was observed in the P. angulata accessions ES and PE and in the P. peruviana accession FE. CMA/DAPI banding revealed three main heterochromatin patterns (CMA⁻/DAPI⁺, CMA⁺/DAPI⁺, and CMA⁺/DAPI⁻), with DAPI⁺ bands predominantly occurring in distal or subterminal chromosomal regions. The number of CMA⁺ blocks varied among species and accessions, with two blocks observed in ES, IX1, and FE, four in PE, and approximately eight in IX3, although some signals could not be precisely counted because of weak fluorescence and chromosome overlap. Flow cytometry showed relative nuclear DNA content values ranging from approximately 45,000–62,000 fluorescence units in the diploid-like profiles of P. angulata and the green-fruited P. ixocarpa accessions IX1 and IX2. In contrast, P. peruviana accessions FE, CO, SC, and SP exhibited values ranging from approximately 94,000–120,000 fluorescence units, approximately twice those observed in the diploid-like profiles and consistent with a tetraploid condition. However, the P. angulata accessions ES and PE showed 2n = 48 chromosomes despite presenting flow-cytometry profiles within the diploid-like range, resulting in a discrepancy between chromosome number and relative nuclear DNA content. Accession IX3 of P. ixocarpa exhibited multiple nuclear populations by flow cytometry, although 2n = 24 chromosomes were observed in its metaphases. Overall, the results demonstrate interspecific and intraspecific variation in heterochromatin organization and nuclear DNA content among the evaluated accessions. The integration of chromosome counting, CMA/DAPI banding, and flow cytometry provided complementary information for the cytogenetic characterization of P. angulata , P. peruviana , and P. ixocarpa , while the discrepancies observed in ES and PE and the multiple nuclear populations detected in IX3 require further investigation.
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