Crop wild relatives (CWRs) are vital sources of variation for genetic improvement, but their populations are few in genebanks, eroded in natural habitats and inadequately characterized. With a view to explore genetic diversity in CWRs of AA genome rice (Oryza sativa L.) species in India, we analyzed 96 accessions of 10 Oryza species by using 17 quantitative traits and 45 microsatellite markers. The morpho-quantitative traits revealed a high extent of phenotypic variation in the germplasm. Diversity index (H’) revealed a high level of within-species variability in O. nivara (H’ = 1.09) and O. rufipogon (H’ = 1.12). Principal component (PC) analysis explained 79.22% variance with five PCs. Among the traits related to phenology, morphology, and yield, days to heading showed strong positive association with days to 50% flowering (r = 0.99). However, filled grains per panicle revealed positive association with spikelet fertility (0.71) but negative with awn length (- 0.58) and panicle bearing tillers (- 0.39). Cluster analysis grouped all the accessions into three major clusters. Microsatellite analysis revealed 676 alleles with 15.02 alleles per locus. High polymorphism information content (PIC = 0.83) and Shannon’s information index (I = 2.31) indicated a high level of genetic variation in the CWRs. Structure analysis revealed four subpopulations; first and second subpopulations comprised only of O. nivara accessions, while the third subpopulation included both O. nivara and O. rufipogon accessions. Population statistics revealed a moderate level of genetic differentiation (F = 0.14), high gene diversity (H = 0.87), and high gene flow (Nm = 1.53) among the subpopulations. We found a high level of molecular variance among the genotypes (70%) and low among populations (11%) and within genotypes (19%). The high level of molecular and morphological variability detected in the germplasm of CWRs could be utilized for the improvement of cultivated rice.© 2023. The Author(s), under exclusive licence to Institute of Plant Genetics Polish Academy of Sciences.