Non-del(5q) transfusion-dependent low/int-1 MDS patients achieve an erythroid response with lenalidomide in 25-30% of cases. Addition of erythropoiesis-stimulating agent could improve the erythroid response rate. The impact of recurrent somatic mutations identified in the diseased clone on response to lenalidomide and the drug effects on clonal evolution remain unknown. We investigated recurrent mutations by next generation sequencing in 94 non-del(5q) MDS patients randomized in the GFM-Len-Epo2008 clinical trial to lenalidomide or lenalidomide + EPO. Clonal evolution was analyzed after 4 cycles of treatment in 42 cases, and re-analyzed at later time points in 18 cases. The fate of clonal architecture of single CD34+CD38- hematopoietic stem cells was also determined in 5 cases. Four genes had a mutation frequency over 10%: SF3B1 (74.5%), TET2 (45.7%), DNMT3A (20.2%) and ASXL1 (19.1%). Analysis of variant allele frequencies indicated a decrease of major mutations in 15/20 responders compared to 10/22 non-responders after 4 cycles. The decrease in the variant allele frequency of major mutations was more significant in responders than in non-responders (P<0.001). Genotyping of single CD34+CD38- cell-derived colonies showed that the decrease in the size of the dominant subclones could be associated with the rise of founding clones or of hematopoietic stem cells devoid of recurrent mutations. These effects remained transient and disease escape was associated with the re-emergence of the initially dominant subclones. In conclusion, we show that, although the drug initially modulates the distribution of subclones, loss of treatment efficacy coincides with the re-expansion of the dominant subclone. The trial is registered to www.clinicaltrials.gov as NCT01718379.