A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.