We produced sequencing data for genome assembly of two Macrostomum species, M. cliftonense and M. hystrix. High molecular weight DNA for PacBio long-read sequencing was extracted from pools of inbred worms for both species by a modified phenol-chloroform protocol. Additional gDNA for Illumina short-read sequencing was extracted from both species using QIAGEN DNEasy kits. For genome annotation we also extracted RNA using Macherey-Nagel NucleoSpin RNA XS columns from pools of adults and hatchlings for both species. These samples were used both for PacBio IseSeq and Illumina TruSeq libraries. We also produced "positional RNA-Seq" data-sets for both species, as well as an improved data-set for the species M. lignano. Finally, we produced population re-sequencing data for population genetic analyses in the form of RNA-Seq data extracted from single worms caught in the wild (for M. cliftonense and M. hystrix) or taken from an outbred laboratory culture. Nuclear genomes were assembled with canu, polished with gcpp and pilon, and annotated with the BRAKER2 pipeline. Mitochondrial genomes were separately assembled with Mitobim and annotated with the MITOS2 webserver.