Three Italian Lolium weed populations, one susceptible and two resistant to diclofop, were characterized by the technique of inter simple sequence repeats (ISSR). The goal of this study was to taxonomically identify these Lolium populations as well as to evaluate evidence for introgression of ISSR fragments from Festuca and the potential role of this introgression in the diclofop response. ISSR analysis confirmed the genomic background of the weed populations to be consistent with that of Lolium. However, the great range of variation in ISSR banding patterns highlighted that the three ryegrass accessions are mixed populations made up of individuals resulting presumably from intrageneric and intergeneric hybridization in the Lolium–Festuca complex. Two Festuca genus-discriminating and 20 Festuca species-discriminating ISSR markers were screened among all the three ryegrass populations. The resistant Tuscania population carried the highest percentage of Festuca genome (16.8%) followed by the resistant Roma (13.6%) and susceptible Vetralla (7.6%) populations. On the basis of these data some influence of Festuca genome in diclofop resistance levels of studied ryegrass populations could be hypothesized.