Regeneration-capable flatworms are attractive models to study the biology of stem cells in vivo. Regeneration-capable flatworm Macrostomum lignano, a genetically tractable model with developed transgenesis methods, is increasingly used to study the biology of stem cells in vivo. In this project we performed comprehensive characterization of gene expression in the proliferating cells of M. lignano, which in adult animals represent somatic stem cells, called neoblasts, and germline cells. We generated a de novo transcriptome assembly of M. lignano based on extensive RNA-seq data and used a combination of two approaches to enrich for genes expressed in either somatic neoblasts, proliferating germline cells or differentiated cells. First, depleting proliferating cells by irradiation. Second, isolating cells in different phases of the cell cycle from adult animals, juveniles, and amputated heads by fluorescence-activated cell sorting. We identified sets of genes enriched in the germline and in somatic neoblasts. The generated M. lignano transcriptome assembly and gene expression signatures of somatic neoblasts and germline cells will be a valuable resource for future molecular studies in M. lignano.