Phenotyping of genetic resources remains the bottleneck in the characterization of genetic resources, since the advent of modern next-generation sequencing technologies has made genotyping much more cost- and time-effective. This article reports on the phenotyping of agriculturally important traits in perennial ryegrass (Lolium perenne). In the present study, water-soluble carbohydrate (WSC), crude protein and dry matter contents were recorded for 1320 individuals, pooled into 132 samples from 33 perennial ryegrass ecotypes and cultivars at five different harvest time points across the 2004 growing season. While, in general, the cultivars had higher WSC contents than the ecotypes, individual ecotypes did show potential to be used in breeding programmes, as they had higher values than all other accessions at particular cutting time points. In correlation analyses, positive relationships were observed between dry matter and glucose contents both early and late in the growing season. Principal components analysis allowed the split either between cultivars and ecotypes or between tetraploid cultivars and the rest of the accessions at four of the five cutting time points. In the analysis of variance, cutting time was the most significant factor influencing the variation in traits.