The aim of this study was to identify and characterize new ISSR markers and their loci in the genome of perennial ryegrass. A subsample of the VrnA F2 mapping family of perennial ryegrass comprising 92 individuals was used to develop a linkage map including inter-simple sequence repeat markers (ISSR). Twelve ISSR fragments out of 29 were mapped to the linkage groups (LG) LG1, LG2, LG4, LG6 and LG7. The total map length was 250.7 cM. Selected ISSR fragments were cloned by transformation into plasmid pTZ57R and sequenced. For four ISSRs, BLAST search revealed a significant similarity to coding regions of known sequences within the current DNA databases. An ISSR fragment of 580 bp, produced by the (GACA) 4 TC primer present on LG6, showed a 95% identity to the Avena sativa L. transposon and repetitive DNA linked to the receptor kinase gene. A 780 bp fragment generated by (TG) 8 RT primer demonstrated a 70% similarity to the Hordeum vulgare germin gene GerA. Inter-SSR mapping will provide useful information for gene targeting, quantitative trait loci mapping and marker-assisted selection in perennial ryegrass. Inter-simple sequence repeat (ISSR) loci mapping in the genome of perennial ryegrass.