Hybridization and introgression can play an important role in genetic differentiation and adaptive evolution of plant species. For example, a conspecific feral species may frequently acquire new alleles from its coexisting crops via introgression. However, little is known about this process. We analyzed 24 weedy rice (Oryza sativa f. spontanea) populations and their coexisting rice cultivars from northern Italy to study their genetic differentiation, outcrossing, and introgression based on microsatellite polymorphisms. A total of 576 maternal plants representing 24 weedy populations were used to estimate their genetic differentiation, and 5,395 progeny (seedlings) derived from 299 families of 15 selected populations were included to measure outcrossing rates. Considerable genetic differentiation (F (st)=0.26) was detected among weedy rice populations, although the differentiation was not associated with the spatial pattern of the populations. Private alleles (28%) were identified in most populations that exhibited a multiple cluster assignments, indicating stronger genetic affinities of some weedy populations. Outcrossing rates were greatly variable and positively correlated (R (2)=0.34, P=0.02) with the private alleles of the corresponding populations. Paternity analysis suggested that ~15% of paternal specific alleles, a considerable portion of which was found to be crop-specific, were acquired from the introgression of the coexisting rice cultivars. Frequent allelic introgression into weedy populations resulting from outcrossing with nearby cultivars determines the private alleles of local feral populations, possibly leading to their genetic differentiation. Introgression from a crop may play an important role in the adaptive evolution of feral populations.