We are developing a linkage map of tetraploid alfalfa (Medicago sativa L.) based on restriction fragment length polymorphism (RFLP) in order to locate genes associated with forage yield, which may facilitate selection and cultivar improvement. Two highly diverse, heterozygous genotypes, WISFAL-6 (M. sativa subsp. falcata) and ABI408 (M. sativa subsp. sativa), were hand-crossed with emasculation to produce an F1 population of 200 individuals. The population was planted in the field at two Iowa locations in 1998 and evaluated in 1999 for dry matter yield. Analysis of yield data confirmed that the F1 population mean was superior to only WISFAL-6 for forage dry matter yield. Concurrently, the parents and F1's were analyzed with RFLP markers using known probes from published diploid and tetraploid maps, cloned genes and ESTs. A total of 85 different probes were screened on the F1 population and 239 segregating loci identified. From these data, 16 preliminary linkage groups were constructed, eight that showed similar markers in both WISFAL-6 and ABI408,five unique to WISFAL-6, and three unique to ABI408. The linkage groups were initially constructed using single dose restriction fragments (SDRF), with double dose restriction fragments (DDRF) and bands expressing segregation distortion added later. Approximately 50% of all bands scored showed segregation distortion. Quantitative trait loci (QTL) analysis was performed and significant QTL with P[Less than or equal to symbol]0.01 were identified. These QTL were compared between environments and harvests. Further development of these two maps and identification of significant QTL will assist in the detection of genes associated with forage yield and other agronomic traits thereby helping to improve the cultivar development process.