Indica and japonica are the two most important subspecies of Asian cultivated rice. Identifying mechanisms responsible for population differentiation in these subspecies is important for indica-japonica hybridization breeding. In this study, subspecies and economic trait differentiation patterns were analyzed using morphological and molecular (InDel and Intron Length Polymorphism) data in F2 and F3 populations derived from indica-japonica hybridization. Populations were grown in Liaoning and Guangdong provinces, with F3 populations generated from F2 populations using bulk harvesting (BM) and single-seed descent methods (SSD). Segregation distortion was detected in F3-BM populations, but not in F3-SSD or in F2 populations. Superior performance was observed with respect to economic traits in Liaoning compared with Guangdong and 1000-grain weight (KW), seed setting rate (SSR), and grain yield per plant (GYP) were significantly correlated with indica and japonica subspecies types. Analysis of molecular and morphological data demonstrated that the environment is the main factor giving rise to population differentiation in indica-japonica hybridization. In addition, we also found that KW, SSR, and GYP are related to subspecies characteristics and kinship, which is possibly a significant factor resulting in economic trait differentiation and determining environmental adaptability. Our study has provided new insights into the process of population differentiation in these subspecies to inform indica-japonica hybridization breeding.