Anthracnose, caused by Colletotrichum trifolii, is a severe disease of alfalfa (Medicago sativa). The RCT1 gene, isolated from the model legume M. truncatula, is a candidate gene to explain genetic variation for anthracnose resistance in alfalfa. A bulk segregant analysis was carried out to test this hypothesis: from each of eight alfalfa varieties, 15 resistant (R) plants and 15 susceptible (S) plants were selected and DNA was extracted. The whole gene including the upstream and downstream regions (a total of 14 kb) was amplified by PCR for each individual and the R and S plants were pooled for each variety. Sequencing was carried out using the next generation sequencer 454 (Roche). The sequence reads, that averaged 295 bp, were assembled to produce consensus sequences that can be considered as alleles. Considering the five exons of the gene, five regions contained clear deletion/insertion polymorphism but these polymorphisms were present in both the R and S pools. Individual genotyping for these indels indicated that different alleles were present but no specific allele was associated with the phenotype. These polymorphic regions in RCT1 seemed not to explain the variation of anthracnose resistance in alfalfa. However, the presence of one rare allele inducing a lack of function was associated with the resistance. A divergent selection for this allele would test its interest in breeding programs.