Angiosperm AGAMOUS-like (AG-like) genes are essential for flower formation. The molecular basis underlying the functions and divergence of four rice AG-like genes that belong to the AG lineage (OsMADS3 and OsMADS58) and AGL11 lineage (OsMADS13 and OsMADS21) is currently poorly understood. In this study, we created AG-like in situ overexpressing (AGisOE) transgenic rice plants for each gene with AG-like fusion with GFP. The AG-like expression domains in AGisOE were found to be similar to those in the wild type, although their expression levels exhibited varying degrees of elevation. In situ overexpression of OsMADS3, OsMADS13, and OsMADS21 perturbed floral robustness and affected flowering time, male fertility, seed-setting rate, and seed-borne fungal growth in different ways. Overall, the fitness of the transgenic plants was reduced in these AGisOEs. Genome-wide characterization of the molecular interactions associated with the AG-like genes revealed that the phenotypic differences observed in the AGisOE lines were well supported by corresponding variations in their direct target genes, putative trans-acting factors, and protein–protein interaction partners. Our results provide new insights into the molecular basis underlying the functional divergence of rice AG-like duplicates in reproductive organs, and reveal the potential significance of variation in gene expression-dosage in plant evolution, the manifestation of new functions, and crop improvement.