Conservation of noble crayfish (Astacus astacus) populations is becoming particularly important since the number of individuals is rapidly declining across the distribution range of the species in Europe. Five crayfish populations in northwestern Poland have been constantly monitored for two decades. However, the genetic structure of these populations has not been analysed, although this information is important to devise effective conservation strategies. Noble crayfish were collected in the autumn of 2014 by scuba diving in Lakes Graniczne, Babinki, Biwakowe, Sęki and Kwisno, all of which are situated in the Bytów Lakeland of northwestern Poland. Genetic diversity of the five populations was assessed based on allele variability in nine SSR regions and six AFLP primer combinations. Microsatellite results analysed with AMOVA showed that the diversity between populations corresponds to 18% of total variability, which was confirmed by similar results obtained using AFLP. Additionally, significant genetic diversity was revealed by high average FST values. All of the studied crayfish populations significantly deviated from the expected Hardy–Weinberg genetic equilibrium and were characterised by negative values of inbreeding coefficient (FIS). The invariably negative inbreeding coefficients (FIS) suggest a low number of mating individuals, a possible consequence of the phenomenon known as genetic bottleneck. However, additional comprehensive analyses are needed to assess the genetic structure, origin and vulnerability of the remaining populations of noble crayfish in the Bytów Lakeland of northwestern Poland, which have high conservation value and are particularly important as a live genetic bank for breeding and restitution purposes.