The genetic variation of Italian ryegrass (Lolium multiflorum Lam.) and westerwolds ryegrass (L. westerwoldicum Breakw.) was investigated in eight tetraploid cultivars originated from Poland. The 337 individual seedlings, cultivated in uniform greenhouse conditions, were tested with horizontal gel electrophoresis according to five enzyme systems: diaforase (DIA), superoxide dismutase (SOD), glutamate oxalacetate transaminase (GOT), peroxidise (PX) and phosphogluco isomerise (PGI), which were used to array allelic diversity at five polymorphic loci. The estimation of genetic diversity in 64 plants of the same cultivars was studied using inter simple sequence repeats (ISSRs) as molecular marker. The genetic variation described by ISSR suggests that the polymorphism detected appears to be poorly informative at the taxonomic level. For statistical analysis, unweighted pair group method with arithmetic mean (UPGMA) based dendrograms and minimum spanning trees were constructed using Nei's distance and Jaccard's similarity coefficient.