The cytophotometric estimation of 4C DNA content, and karyotypic and somatic chromosome number analyses were carried out in 30 genotypes comprising seven cultivars and 23 advanced breeding lines of Solanum tuberosum. Detailed karyotype analysis revealed genotype specific chromosomal characteristics and structural alterations in chromosomes of the genome, with a rare phenomenon of aneusomatic (2n = 4x + 2 = 50) condition in cv.K. Chandramukhi. The origin of this variation could be attributed to mitotic non-disjunction in the shoots giving rise to aneusomatic roots. Highly significant variations in the genome length, volume and total form percentage were noted at the cultivar level. The total chromosome length varied from 84.56 μm in cv.K. Pukhraj to 127.62 μm in MS/89-60, with an average value of 100.94 μm ± 1.82. Total chromosome volume varied from 57.22 μm3 in MS/92-1090 to 132.64 μm3 in JW-160. Significant variations in the 4C DNA content (7.28–15.83 pg) were recorded at the cultivar level, with an exceptionally high DNA content (22.24 pg) in cv.K. Chandramukhi. This could be due to the aneusomatic condition of this genotype. Correlation studies revealed interdependence between the chromosomal and nuclear parameters of the genotypes. Structural alterations in the chromosomes, as well as loss or addition of highly repetitive sequences in the genome, caused variations in DNA content at the cultivar level. Variations in genomic structure and nuclear DNA content of the 48-chromosome genotypes suggest a genetic drift during microevolution, leading to the development of new cultivars.