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The goldenberry, Physalis peruviana L., is an important fruit in Colombia due to its export value and nutritional quality. However, commercial crops face challenges of fruit heterogeneity and the presence of diseases that reduce fruit yield and quality. These drawbacks could be handled through different breeding methods to develop uniform cultivars, for example, through anther culture, which is used to rapidly produce homozygous lines. However, the ploidy level may change when using this technique. Therefore, in this study, the level of ploidy of parental and the plants obtained by anther culture was determined by cytogenetics, flow cytometry, and single-simple repeat or microsatellites (SSR).Additionally, the homozygous condition of obtained plants and the degree of heterozygosity of parental plants were evaluated using SSR. Cytogenetic analysis showed parental plants with 48 chromosomes and anther culture generated plants with 24, 32 and 48 chromosomes, and mixoploids and average nuclear DNA content between 5.04 and 20.08 pg. Diploid, tetraploid, hexaploidy, and octoploid plants were identified by flow cytometry; the highest levels of ploidy (6x and 8x) correspond to mixoploid plants found by cytogenetics. The SSRs did not allow identifying ploidy due to the lack of a particular band pattern, however, they showed heterozygosity in most of the plants obtained by anther culture. It was concluded that the anther culture modified the ploidy level with respect to the parental plants, and that flow cytometry is efficient, precise, and less laborious, compared to cytogenetics, to determine the ploidy level of goldenberry in the laboratory.