Double ‘free-hybrids’ (DH) in alfalfa were obtained by crossing in a diallelic scheme, six multiplied simple hybrids (SH) derived from four partly inbred (S2) lines. Analysis of the specific combining ability demonstrated that the main source of variation was for dry matter yield (DMY) in DHs and supported heterosis values of DHs versus the best parent of an average +45% (ranging from +5 to +76%). Investigation at the molecular level was carried out by analysis of simple sequence repeat markers on the six parental SHs and 15 DH progenies and by comparison of gene expression profiles using microarrays of a single DH line to its parental lines. The variation of heterozygosity estimates of the DHs explained a small part (about 20%) of their variation in DMY, while the number of alleles was significantly related to DM performance (r = 0.61; P < 0.05). The microarray analysis identified genes with both significant additive and non-additive levels of expression in the hybrid compared with the parents. The majority of the variation in gene expression was additive (87%), but among the genes with a non-additive pattern of expression, the greater proportion of probe sets (86%) fell outside the parental range. Gene ontology analysis of these genes revealed the presence of a number of terms related to metabolism and genetic information processing.