Collection and characterisation of genetic resources are required for the development of new cultivars. We analysed genetic diversity among 18 non-dormant lucerne (alfalfa, Medicago sativa L.) accessions including ten local ecotypes and eight introduced accessions at morphological and molecular levels using sequence-related amplified polymorphism (SRAP) primers. Standardised canonical discrimination functions for the investigated morphological traits showed that the first function (explaining 75% of total variability among accessions) was strongly influenced by leaflet shape, stipule shape, and the peduncle : petiole length ratio. The 16 SRAP primer pair combinations generated 677 differently sized SRAP fragments (peaks), of which 665 (98.3%) were polymorphic across all 18 accessions. We detected high levels of polymorphism (average polymorphic information content value = 0.96, average of 42.3 polymorphic fragments per primer pair). Based on morphological and SRAP data, local accessions tended to group together in the same cluster or formed individual clusters. Clusters of local accessions at high similarity sometimes correlated with their collection site (Qasemi-2 and Qasemi-3) on molecular analysis. Results of cluster analysis based on SRAP showed no significant correlation with morphological characters based on the Mantel test (r = 0.04).This inconsistent clustering of accessions could be due to the allelic variation (presumably) in a small number of genes (24 traits) contributing to morphological characterisation, while the 677 SRAP fragments (loci) are assumed to be relatively widely distributed across the genome. The wide geographical distribution of lucerne populations across different environments may provide good genetic resources for breeding purposes. SRAP analysis was effective to study genetic variability of non-dormant lucerne. This information will be helpful in assessing selections for lucerne breeding programs to develop new cultivars adapted to harsh environmental conditions.