Angiosperm AGAMOUS-like (AG-like) genes are essential for flower formation. The molecular basis underlying the functionsdivergence of four rice AG-like genes that belong to the AG lineage (OsMADS3OsMADS58)AGL11 lineage (OsMADS13OsMADS21) 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,OsMADS21 perturbed floral robustnessaffected flowering time, male fertility, seed-setting rate,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,protein–protein interaction partners. Our results provide new insightsthe molecular basis underlying the functional divergence of rice AG-like duplicates in reproductive organs,reveal the potential significance of variation in gene expression-dosage in plant evolution, the manifestation of new functions,crop improvement.