Abstract: Four populations of Norway spruce (Picea abies Karst.) were screened using nine nuclear microsatellite markers (three trinucleotides and six dinucleotides) and four chloroplast markers (all mononucleotides). Marker classes were compared for their variability, mutation rate and ability to detect differentiation between stands. Dinucleotide markers proved to be the most variable group and chloroplast stretches the least variable, with differences in mutation rate between the former and the latter spanning over two orders of magnitude. Variability correlated to the number of repeats but not to the absolute length of the microsatellite region. The different marker classes were combined with two different measures of genetic distance in order to investigate the performance of markers and evolutionary models for the study of genetic variation in natural populations of Norway spruce. Weir and Cockeram's F-ST generally performed better in this clear-cut, four-population model study. Chloroplast haplotypes turned out to be the most sensitive marker system, being able to differentiate populations and to detect differences in genetic variability between sub-regions.