The genus Lolium (ryegrasses) exhibits substantial variation between species for annual-perennial growth habit. The genetic bases of this trait, and other characters that are differentiated between taxa, have been investigated through molecular genetic marker-based mapping of an interspecific mapping population derived from pair-wise crossing of single genotypes from Lincoln, a long-lived cultivar of perennial ryegrass, and Andrea, a cultivar of annual-type Italian ryegrass. The Andrea1246 and Lincoln1133 parental maps contained 122 loci on eight linkage groups (LGs), and 169 loci on seven LGs, respectively. A total of 10 phenotypic traits were measured, including annuality-perenniality index, date of head emergence, number of spikes per plant, number of spikelets per spike, number of floret per spike, flag leaf length, flag leaf width, spike length, stem length, and extent of regrowth. A total of 31 putative quantitative trait loci (QTLs) were detected. Regions of significance were identified on Andrea1246 LGs 1, 2, 3 and 6. An annuality-perenniality index QTL on LG2 accounted for ~30% of trait-specific phenotypic variance (Vp). In addition, LG2 contained coincident QTLs for the number of spikes per plant and head emergence date traits. The Lincoln1133 genetic map displayed QTL-containing regions of significance on LGs 1, 4, 5 and 7, accounting individually for 10–22% of Vp. QTLs identified in this study provide potential targets for ryegrass breeding in order to improve vegetative yield, persistence and seed yield.