Microalgae remain an exciting target for biotechnology as they offer a largely unexploited reservoir of novel and valuable bioactive compounds. Strain improvement programs are an expanding research field aiming to multiply microalgal potential. This study evaluates the genetic diversity created in populations of the diatom Phaeodactylum tricornutum subjected to random mutagenesis. We explored the genetic diversity using genotyping-by-sequencing (GBS) to estimate and compare the impact of the most common chemical mutagen (ethyl methanesulfonate, EMS). Five microalga populations obtained following EMS treatment had survival rates between 1 and 98 %. High genetic diversity was obtained for only one of these P. tricornutum populations, with a survival rate close to 30 %.