Eleven laboratories have collaborated to study chloroplast DNA (cpDNA) variation in black poplar (Populus nigra L.) across Europe in order to improve our understanding of the location of glacial refugia and the subsequent postglacial routes of recolonisation. A common analysis based on the restricted fragments produced by five primer pairs was used to determine the cpDNA haplotype of 637 samples obtained from genebank collections established in nine European countries. Haplotype 2 was particularly common and was found in 46% of the non-hybrid samples. A total of 81 non-hybrid chloroplast variants were detected. Three haplotypes (from four trees believed to originate from Eastern Europe) clustered together and were very different from the rest of the samples. The remaining samples were divided into two groups, one of which had a largely eastern distribution and samples from the other group were mostly located in the west. This, along with the fact that Spain in the southwest and Austria and Italy in the southeast had high diversity, suggest that there were ice age refugia of black poplar in both southwestern (Spain) and southeastern Europe (Italy and/or Balkan). Results also indicate that the Pyrenees formed a significant barrier, since only 7 of the 45 haplotypes in Spain exist elsewhere in Europe. © 2004 Elsevier B.V. All rights reserved.