The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H(T) = 0.586, and this diversity was distributed predominantly among rather than within populations (F(ST(M)) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada. Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a; while populations in northern Fennoscandia were fixed for mitotype b. The isolated Scottish populations were predominantly of mitotype a, but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H(T) = 0.120) and genetic differentiation among populations (F(ST(M)) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.