The nutritional quality of lucerne (alfalfa, Medicago sativa L.) plants correlates positively with the presence of multifoliolate (MF) leaves. Using phenotypic recurrent selection, we developed populations with an increased percentage of MF expression from 6.7% in the original population (C0) to 77.7% in the fourth cycle (C4). The effect of selection on genetic diversity within and among populations was evaluated. The populations C0 and C4 were represented by 40 plants genotyped by using 25 simple sequence repeats (SSR). The number of alleles per locus was large in both C0 and C4, averaging 6.28. The within-population genetic diversity (H_E) overall estimation was 0.723 for C0 and 0.726 for C4, the absence of significant difference between the two populations indicating that the genetic diversity was as large in C4 as in C0. The Nei’s population differentiation (G_ST) overall estimation was 0.013, meaning that only 1.3% of the total genetic diversity was between populations and 98.7% was within populations. An efficient selection process was conducted without any increase in inbreeding or genetic drift.