In the present study, a set of chromosome segment introgression lines (CSILs) using Gossypium hirsutum L. TM-1 as the recipient parent and G. barbadense Hai7124 as the donor parent were used to explore the genetic basis of heterosis for interspecific hybrids. Two sets of F₁ populations individually derived from CSILs crossing with both parents were configured to investigate heterotic loci (HL) and substitution effect loci (SL). A total of 58 HL and 39 SL were identified in 3 years. One stable HL, hLP-A4-3, could be detected in all 3 years. Three HLs, hBS-A8-1, hLP-D6-1, and hSI-D7-11, could be detected in 2 years. Four SLs, sBS-D7-1, sLP-A8-1, sLP-D7-1, and sLP-D12-1, could be detected in 2 years. HL and SL tended to be distributed in some HL-rich chromosome segments with close positions. Compared with QTL detected in a former study, HL showed little overlap with QTL, indicating that trait phenotype and heterosis might be controlled by different sets of loci. All three forms of genetic effects (partial-, full-, over-dominant) were identified, while the over-dominant effect made the main contribution to heterosis. These results may help lay the foundation for clarifying the heredity mechanism of heterosis in cotton.