Lolium perenne (ryegrass) is a species of major economic importance due to its multiple uses such as forage, turf, and cover crop and its potential use as a feedstock for bioenergy. Ryegrass occurs naturally as a diploid (2n=14). However, several autotretraploid cultivars were obtained via colchicination. Ryegrass displays a two-locus gametophytic self-incompatibility system (SI) that remains intact even at the tetraploid level. There are two plausible models for SI at the tetraploid level, Model I in which only one different allele in the pollen grain from the female at either S or Z locus makes pollen compatible. Model II states that any S-Z combination in the pollen grain matching the female would make pollen incompatible. The goals of this study are i) to evaluate which of the two models better explains SI in our bi-parental autotetraploid ryegrass population; ii) to provide evidence that SI maintenance was not specific to genotypes used in previous studies; and iii) to evaluate the sensitivity and efficacy of HRM genotyping in polyploid species. The present study used 43 progeny of a cross between two autotetraploids and characterized them with three HRM-based markers that were reported to co-segregate with Z. The confirmation of the underlying model for SI in autotetraploids in combination with the use of markers as well as automated software for pollen compatibility estimation such as PollenCALC can be of great use to perform controlled crosses in tetraploid ryegrass.