Alfalfa (Medicago sativa L.) is one of the most important leguminous forage crops around the world. Improving alfalfa yield and persistency is a continual goal for plant breeders. Cultivated alfalfa is an outcrossing tetrasomic tetraploid, which means that a significant reservoir of deleterious (or unfavorable) alleles can be maintained in breeding populations, limiting progress for important quantitatively inherited traits like yield. Inbreeding depression, occurring due to the umasking of these alleles, limits the development of inbred lines, which would be useful for breeding and mapping purposes. The objective of this experiment is to use anther and/or microspore culture to recover alfalfa haploids (1x), and then to double their chromosomes to produce diploid and tetraploid homozygous lines, thereby accelerating cultivar improvement. Our strategy is to develop an anther culture protocol using tetraploid genotypes in order to produce 2x dihaploids. Once this protocol is developed, we will attempt to isolate true (1x) haploids. We are using different combinations of alfalfa genotype, culture medium, and stage of pollen development in which the anthers are isolated to optimize the system to make haploid alfalfa. Several parental lines used for mapping populations and a group of culture media are being tested. Flower buds at different stages of pollen development and with various pollen sizes are being used for callus induction. Calli from anther culture will be regenerated into shoots and ploidy will be determined. We will discuss our progress to date and plans for the future.