Self-incompatibility (SI) is a mechanism that prevents self-pollination and inbreeding in many flowering plant species. SI in Lolium perenne is controlled by two multi-allelic loci, S and Z. SI has important consequences for L. perenne breeding as it prevents the efficient production of inbred lines and hybrids. In this study, the cDNA-AFLP approach was used to identify key components of the SI response, S and Z, as well as genes involved in the signaling cascade triggered by a SI response in L. perenne. A total of 169 transcripts displaying an allele-specific expression profile were identified. Several of these transcripts displayed homology to genes involved in general cellular functions. In other cases, interesting homologies to proteins, such as kinases, known to be involved in SI in other plant families were found. A genome-wide expression analysis, on the other hand, allowed us to identify 515 transcript derived fragments as putatively related to SI in L. perenne. All the expression profiles were quantified using AFLP-QuantarPro (TM) and clustered using hierarchical and AQBC clustering methods. A subset of these genes was selected for sequencing and assigned into 10 functional categories. Homologies were found to proteins known to be involved in fertilization and SI processes in other plant families such as ubiquitin-related and calcium-related proteins.