Data from the pollen record and from two forms of nuclear genetic markers suggest that present-day populations of Scots pine (Pinus sylvestris L.) in Scotland were derived from more than one refugium after glaciation. In order to clarify this issue, genetic variation for maternally inherited mitochondrial DNA (mtDNA) was studied in 466 trees sampled from 20 natural populations in Scotland. A homologous probe for the cox1 mitochondrial gene of P. sylvestris was constructed and used to detect mtDNA RFLP variation. Two common (a and b) and one rare RFLP variant (c) were distinguished. Evidence from segregation patterns of variants within a polymorphic population was consistent with maternal inheritance of the RFLP variation. A survey of Scottish populations indicates that mitotype a is present at all sites, but that mitotype b is confined to three western populations. Genetic differentiation for mtDNA, which migrates solely by seed is much greater (F(ST(m))= 0.370) than for nuclear markers (F(ST(b))= 0.028) which are dispersed by both pollen and seed. The geographical distribution of mitotype b in western Scotland, and its absence from populations in northern France and Germany, suggest that P. sylvestris has been derived not only from continental Europe via England, but also by migration from a western refugium, probably in Ireland or western France.