Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] is an economically valuable non-native tree species in Germany and is considered very promising in view of global climate change. Therefore, the genetic characterization of Douglas-fir populations and seed stands in Germany is essential. We studied coastal and interior Douglas-fir varieties, both present in Germany, by using eleven isoenzyme and four microsatellite loci. By analyzing eight reference populations of known origin we were able to identify the two varieties on the population level using Bayesian and distance based methods. Seven populations present in Bavaria were then successfully assigned to one of the two varieties. Within varieties we found stronger grouping within the interior variety than within the coastal one. Despite lower differences within coastal Douglas-fir we have first indications for the origin of two populations. For two Bavarian populations, natural regeneration was included and genetic data revealed no significant genetic difference between adults and offspring. The parentage analysis for one of the studied stands revealed that a large proportion of adults took part in the reproduction, but some trees were more successful than others in transferring their genes to the next generation. Our study was able to improve variety identification of Douglas-fir using isoenzyme markers and nuclear microsatellites and study reproductive patterns, both are important issues for the management of Douglas-fir stands in Bavaria.