A potential strategy to decrease the levels of self-seed production during the seed increase stages of alfalfa (Medicago sativa L.) synthetic cultivar development is selection for decreased self-fertility. The underlying genetics of this trait have not been elucidated and, therefore, a study was designed to identify genetic determinants of alfalfa self-fertility. An F1 mapping population developed by crossing parents representing Medicago sativa subsp. falcata and M. sativa subsp. sativa was characterized for self-fertility in a greenhouse during the winter of 1999. Traits representing self-fertility were measured and then mapped to the resulting genetic maps of both parents. Heritability values for each trait were high, suggesting genetic factors are important for their expression. Quantitative trait loci for self-fertility traits were identified on linkage groups (LGs) 2 and 4 from the falcata parent and LGs 4 and 8 of the sativa parent.