We have constructed RAPD-based linkage maps for an interspecific cross between two species of the genus Populus (P. adenopoda and P. alba), based on a double pseudo-test-cross strategy. Of a total of 360 polymorphic fragments scored, 290 showed a test-cross configuration, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. In the female parent, P. adenopoda, 82 markers were grouped in 19 different linkage groups (553 cM), whereas in the male parent P. alba, 197 markers established a much more complete framework map with an observed genome length of 2300 cM covering 87% of the total P. alba genome. The larger number of test-cross markers detected for the P. alba parent than for the P. adenopoda parent might be due to a higher level of heterozygosity in the former than in the latter. In this study, we detected only a small percentage (2%) of the intercross dominant markers heterozygous in both parents and segregating 3:1 in the progeny. The further focus in this mapping study should be on the identification of more intercross markers, to align the two parent-specific maps into a consensus map for mapping important genes causing species differentiation during long evolutionary divergences.