Nucleotide sequence data (cytochrome b) and ISSR genomic fingerprints were used to analyse the genetic variation and population differentiation in Thorn-tailed Rayadito, a widespread Patagonian forest bird. We included samples from eight populations of Thorn-tailed Rayadito covering most of the distribution range of the species: from fragmented patches of Olivillo forest in northern Chile to Isla Navarino forests in the extreme south of South America. Low levels of genetic diversity were found among populations, with a large within-population molecular variance indicating high levels of gene flow. The multivariate and cluster analyses based on ISSR markers show that the subspecies bullocki (from Mocha Island) differs significantly from all other populations. The subspecies fulva (Chiloé Island) shows less differentiation than bullocki, sharing several alleles with continental populations. Bayesian analyses suggest that the Mocha Island population contributes most to the total genetic diversity observed in the species. Mantel tests revealed no significant correlation between geographical distance and pairwise genetic distance and cytochrome b sequence analyses failed to detect differentiation among subspecies. Mocha Island might have been a palaeorefuge and this population may have diversified by genetic drift after the last glacial maximum. There is also the possibility of a postglacial colonization of the Thorn-tailed Rayadito from an austral palaeorefugium, supporting a multiple refugia hypothesis. This study illustrates the usefulness of the rarely used ISSR genomic fingerprint method in avian phylogeography.