Background: Utilization of heterosis has greatly enhanced the productivity of many crops worldwide. Understanding the potential molecular mechanism about how hybridization in cotton produces superior yield is critical for efficient plant breeding. Results: With the whole-genome RNA sequencing, here, high, medium, and low hybrids varying in level of yield heterosis were screened based on different years and locations field experimentation. Phenotypically, high Department showed a mean of 14% more seed cotton yield than its better parent. A total of 63 samples comprised of different squaring stage tissues of three hybrids and four their inbred parents were used to perform transcriptomic analysis. A comparison of transcriptomic differences in each hybrid parent triad revealed a higher percentage of differentially expressed genes (DEGs) in each tissue. Expression level dominance analysis exposed the majority of hybrids DEGs followed parent like expressions. Functional annotations identified an array of DEGs involved in ATP and protein binding, membrane, cell wall, mitochondrion, and protein phosphorylation. Starch and sucrose metabolism and plant hormone signal transduction pathways were most enriched in each hybrid. Further, these two pathways had most mapped DEGs on known seed cotton yield QTLs. Integration of transcriptome, QTLs, and gene co-expression network analysis raveled genes GhBZR1, GhASK8, At3g43860, GhGBSS1, GhAPL2, GhMPK4, GhPHO1, GhJAZ10, and GhCRR21 displayed a complex regulatory network of many interconnected genes. qRT-PCR of these DEGs was performed to ensure the accuracy of RNA-Seq data. Conclusions: Through genome-wide comparative transcriptome analysis, the current study provides novel insights about phenomics and genomics of heterosis in upland cotton. Our results and data resources will be useful for dissecting the molecular mechanism of yield heterosis in cotton.