Mashua (Tropaeolum tuberosum) is a vegetatively propagated tuber crop cultivated in the Andes. This study aims to determine mashua's genetic diversity and structure, which have never been investigated until now. Sixty-eight accessions collected in nine departments of Peru were analyzed with 17 intersimple sequence repeat primers. Three multivariate analysis methods (multiple correspondence analysis, clustering analysis, and assignment method) were used to investigate the genetic structure of the species. A total of 193 reliable markers were generated, of which 169 (88%) were polymorphic. Except for four accessions, the results of the three methods were in perfect agreement. They established the existence of three groups of accessions corresponding to the northern, central, and southeastern regions of Peru and showed a genetic structure clearly influenced by geographical provenance. The average genetic distance over all the accessions (Jaccard's coefficient) showed a moderate level of diversity (0:35 ± 0:05). It may be explained by the reproductive and farming characteristics of the species. The methodology used here to study mashua has revealed valuable and reliable information on its genetic diversity and structure, which should be investigated more extensively along the Andes to establish appropriate conservation strategies. © 2008 by The University of Chicago. All rights reserved.