Sorghum (Sorghum bicolor (L.) Moench), the world's fifth most economically important cereal crop, is renowned for its exceptional drought tolerance and water-use efficiency. Hybrid breeding, a cornerstone strategy for yield enhancement through the exploitation of heterosis, has been extensively employed in sorghum improvement. Despite its agricultural importance, the molecular mechanisms driving heterosis remain largely unresolved. To address this knowledge gap, we conducted whole-genome resequencing of two elite parental lines, AJ2055 and RN133, which have contributed to the development of over 30 commercial hybrids. Our objectives were to delineate genomic variations associated with heterosis performance and elucidate their functional implications in yield-related traits. Genomic analyses revealed substantial divergence between the two parental lines: AJ2055 harbored 2,961,777 single nucleotide polymorphisms (SNPs) and 474,247 insertions and deletions (InDels), whereas RN133 exhibited 54,724 structural variations (SVs) and 36,515 copy number variations (CNVs). Chromosomes 5 and 10 showed the highest SNP densities, with distinct spatial distribution patterns distinguishing the two lines. Notably, RN133 exhibited a broader and more diverse array of genetic variations compared to AJ2055, potentially underlying its superior performance in hybrid combinations. Functional annotation of the variant genes revealed their involvement in essential metabolic pathways, including carbon metabolism, starch and sucrose metabolism, and hormone signaling-related pathways. Key genes encoding enzymes such as phosphoenolpyruvate carboxylase, 1,4-alpha-glucan-branching enzyme, and cytochrome P450 were found to harbor non-synonymous mutations, potentially influencing yield-related traits and stress adaptation. These findings provide valuable insights into the genetic basis of heterosis and lay a foundation for the development of molecular markers for sorghum breeding. In conclusion, this study highlights the distinct patterns of genomic variation between AJ2055 and RN133 and their potential candidate genes and pathways involved in heterosis. The identification of yield-related genes and pathways offers a theoretical basis for future research on sorghum genetic improvement and hybrid breeding. Through the integration of multi-omics approaches and advanced breeding technologies, these findings will contribute to the development of high-yielding sorghum varieties.