Annual ryegrass (Lolium multiflorum Lamarck) is planted annually on more than 1 million ha of pastures in the southeastern United States. The exact ploidy level and genome size of the annual ryegrass germplasm collection (USDA-ARS, Germplasm Resources Information Network - GRIN) are unknown and the future use of this germplasm relies on their determination. The study was conducted in order to: 1) estimate the ploidy level and determine the DNA content of 139 accessions from the GRIN collection, 15 experimental lines and 13 commercial cultivars through flow cytometry, and 2) identify morphological and agronomic characteristics that could be used to differentiate diploid from tetraploid annual ryegrass. A total of 140 diploid and 27 tetraploid lines were successfully identified through flow cytometry. The average 2C DNA content was 6.13±0.36 pg for the diploid lines and 12.30±0.83 pg for the tetraploids. Diploid and tetraploid annual ryegrass showed significant differences for morphological and agronomic variables such as seed size, leaf width and length, guard cell size, plant height, tiller size, plant biomass and seed head length, and all variables were significantly correlated with DNA content. The use of morphological and agronomic variables may guide the determination of ploidy when flow cytometry is not available. Ploidy level and genome size information will benefit those that request accessions from the GRIN system for specific studies at the diploid or tetraploid level in the species.