Polyploidy and ecotypic differentiation are important aspects of plant evolution. The present study has applied molecular methods to investigate the polyploid origin and ecotypic differentiation of the tetraploid Cochlearia officinalis in Northern Scandinavia (comprising three subspecies/ecotypes), in the context of related species in section Cochlearia. The genetic results from six microsatellites markers (cross-amplification of markers developed for other species in Brassicaceae) and thousands of single nucleotide polymorphisms (SNPs) retrieved from restriction-site associated DNA sequencing (RAD-seq) are largely congruent, although displaying different levels of resolution. The species investigated, representing different ploidal levels from diploid to octoploid (C. pyrenaica, C. aestuaria, C. groenlandica, C. officinalis and C. anglica, as well as the hybrid C. x hollandica), seems to be genetically closely related, reflecting a section consisting of recently derived species where gene flow may occur both between ecotypes and across ploidal level. The genetic structure detected in Cochlearia officinalis in Northern Scandinavia can only to some extent be explained by the different subspecies/ecotypes per se. Both geographical distance and population affiliation are also to a large extent responsible for the patterns found. Cochlearia officinalis ssp. integrifolia (the spring ecotype) is the most distinct of the three subspecies, whereas the two other subspecies, C. officinalis ssp. norvegica (the estuary ecotype) and C. officinalis ssp. officinalis (the beach ecotype) are more genetically overlapping. A single (auto)polyploidization event resulting in C. officinalis in Northern Scandinavia is proposed, based on the genetic data. The subspecies/ecotypes of C. officinalis constitute a single group, distinguished clearly from other species and ploidal levels, based on the high-resolution RAD-seq data. The events leading to ecotypic differentiation in C. officinalis within Northern Scandinavia are discussed, but cannot be fully elucidated by the genetic results in the present study. Most evidence (including previous studies on morphology and physiology) seems to support a single ecotypic differentiation event with subsequent dispersal for the spring ecotype, whereas the ecotypic differentiation of the coastal subspecies (the estuary and the beach ecotypes) is less evident from the available data. Both repeated ecotypic differentiation, as well as a single ecotypic differentiation can be argued for.