Improvement of local maize varieties (Zea mays L.) in Indonesia has not been developed yet, one of them is Pulut Sulawesi. Zea mays L. Var. Pulut has low yield productivity around 2-2,5 tons/Ha. In meanwhile, Zea mays L. Var. Black Aztec has higher productivity around 6 tons/Ha, resistance to pests and diseases, and has high anthocyanin content which was beneficial to human health. The first step in a plant breeding program is to study the chromosomes characterization. The aim of the study was to determine the karyotype formula and the value of the R difference among the two varieties. Chromosome staining and microscopic preparation used in this study were Squashing method. Then, measured by using Image Raster 3.0. The results revealed that both varieties have similar karyotype formula (2n = 20m). Based on the R-value of the two kinds of Maize (R= 0,162 ≤ 0,27), it indicated that two kinds of Maize were considered to be originated from similar species.