In this study, each of 60 rRNA internal transcribed spacer (ITS) 1 and ITS2 sequences was determined from 44 individuals of 14 morphologically identified New World sand fly Lutzomyia species in Ecuador, and their interspecies and intraspecies genetic diversity was compared. Distinguishing between related species based on the ITS1 sequence was difficult because of variability, while the genetic diversity of ITS2 was distinct even among closely related species. Further, an assessment of intraspecies ITS sequence diversity in the subgenus Helcocyrtomyia revealed no correlation between sequence variation and geographic distribution. The results strongly suggested ITS2 to be a more suitable marker than ITS1 for the taxonomic analysis of Lutzomyia species including closely related species. Moreover, neither ITS sequence may be useful for the analysis of population structures in Lutzomyia species. © 2009 Elsevier B.V. All rights reserved.