High non-structural carbohydrates (NSC) concentration in forages improves N utilization by ruminants and could significantly increase the ethanol conversion rates from alfalfa (Medicago sativa L.) biomass. Two populations (NSC+ and NSC-) were obtained by intercrossing 10 genotypes selected for high or low NSC concentrations among 500 genotypes of the cultivar AC Caribou. A third population (NSC0) was generated by intercrossing 10 randomly chosen genotypes. We used the sequence-related amplified polymorphism (SRAP) technique to uncover DNA variations associated with NSC accumulation. Biochemical analyses confirmed that the NSC+ population had higher NSC concentrations than the NSC- population. An initial screening of 42 SRAP forward and reverse primer combinations with bulk segregant analysis (BSA) of DNA samples (96 genoptypes) from each population was performed. Five polymorphic bands that varied in intensity between high and low NSC were identified. Both positive and negative polymorphisms were detected. Assessment of genotypic occurrence of a fragment uncovered with the Me2/R7 primer pair confirmed that polymorphism detected with BSA reflects changes in its frequency between populations. Sequence analysis of the purified Me2/R7 fragment did not reveal any significant homology with sequences in databases. These results indicate that regions associated with NSC accumulation can be identified within the alfalfa genome. Future studies will assess the value of these polymorphisms in marker-assisted breeding.