Suitable genetic markers for population studies in sponges are necessary to further our understanding of biodiversity and dispersal patterns, and contribute to conservation efforts. Due to the slow mitochondrial substitution rates in demosponges, nuclear introns are among the preferable markers for phylogeographic studies, but so far only the second intron of the ATP synthetase beta subunit-gene (ATPSβ) has been successfully established. In the present study, we analyse the intron of the Lysidyl Aminoacyl Transfer RNA Synthetase (LTRS), another potential marker to study demosponge intraspecific relationships, on samples of Neopetrosia chaliniformis from various locations in the Indo-Pacific and compare its variation with a mitochondrial marker (CO2). LTRS recovers several reciprocal monophyletic groups among the Indo-Pacific N. chaliniformis and provides a potential alternative to ATPSβ.