Intra- and inter-sample similarities for four populations of the annual grass weed Phalaris minor from Haryana state, India, were examined using inter-simple sequence repeat (ISSR) DNA markers. Levels of polymorphism within and between populations were low in comparison with values reported from other grassy weed species. Analysis of inter-population similarities allowed a partial differentiation of the four populations and of pairs of populations classified by cropping system. Analysis of the intra-population similarity data showed a weak but consistent and statistically significant negative correlation between the molecular similarity of seedlings and the physical distance between their mother plants over distances up to 40 m (the maximum separation tested) in all four populations. The consistency of the observed relationship between molecular similarity and physical separation, and the differences in cultivation practices at the four sites, suggested that the relationship may be a result of localized out-crossing, rather than an effect of localized seed rain. The results of the analyses are discussed in relation to the potential for evolution of multiple traits in the weed in response to changes in the wheat production system in the region.