<p>Cytotype diversity plays a decisive role in shaping the genetic architecture of yield and quality traits in the highly polyploid crop Sugarcane. In this study, 48 interspecific crosses derived from cultivars (females) and cytotypes (males) of <i>Saccharum spontaneum</i> L. differing in ploidy levels (2<i>n</i> = 40–112 chromosomes) were evaluated using REML-based combining ability analysis. Significant genetic variability was detected for all traits. Additive variance predominated for number of millable canes (NMC; <i>σ</i>²<i>F</i> = 242.75, <i>σ</i>²<i>M</i> = 201.09, Baker’s ratio = 0.89, <i>H</i>² = 0.96), plant height (Baker’s ratio = 0.98), and cane diameter (Baker’s ratio = 0.91, <i>H</i>² = 0.91), indicating strong transmissible genetic effects. In contrast, internode length was largely governed by non-additive gene action (<i>σ</i>²SCA = 3.24; Baker’s ratio = 0.19). Brix % exhibited mixed inheritance with substantial GCA (male = 21.80) and SCA (20.38) variances (Baker’s ratio = 0.53; <i>H</i>² = 0.93). Cytotypes differed markedly in combining ability where moderate-ploidy cytotypes (64–80 chromosomes) showed consistent positive GCA effects for NMC and plant height, suggesting optimal gene dosage balance at intermediate ploidy levels. High-ploidy cytotypes such as 2<i>n</i> = 96 and 2<i>n</i> = 112 displayed trait-specific superiority, particularly for Brix %, supporting allele dosage effects in sucrose accumulation. However, cytotype-specific uniform superiority across all traits could not be observed, which confirms&#xa0;that gene balance rather than absolute chromosome number governs quantitative performance. Clones with different cytotypes, viz., SES 590 (2<i>n</i> = 64), SES 574 (2<i>n</i> = 80), SES 404 (2<i>n</i> = 64), SH 216 (2<i>n</i> = 72), and IND 84-415 (2<i>n</i> = 80) were found superior general combiners. SCA analysis identified highly promising crosses, including CoH114 × SH 216 (NMC SCA = 5.654**) and Co 1148 × SES 404 (Cane&#xa0;diameter SCA = 5.030**), demonstrating strong heterotic interactions between specific cytotype–cultivar combinations. Multivariate analyses (PCA explained 76.7% of the variation) further revealed clear divergence among crosses derived from contrasting cytotypes. Natural intraspecific hybridization and selfing within the cytotype caused extensive morphotypes which caused differential variation in the families of the same cytotype. Overall, cytotypes function as key genomic regulators influencing additive variance, heterosis expression, and trait trade-offs, underscoring their strategic importance in sugarcane improvement programs. Strategic deployment of complementary cytotypes with favorable combining ability will be essential for achieving balanced improvement of cane yield and sucrose content with climate resilience in sugarcane breeding programs.</p>