As a basis for comparative studies in the family Cicindelidae (tiger beetles), a phylogenetic hypothesis for major groups was established based on molecular data, focusing on the taxa of the New World. Two markers, mitochondrial 16S rRNA and nuclear 18S rRNA, were sequenced for a total of 912 nucleotides and 30 taxa. All Cicindelidae were inferred to be a monophyletic group by the presence of a segment in the 5' region of the 18S rDNA which is predicted to fold into a single stem-loop structure not present in the outgroup. The total number of variable and potentially informative positions was smaller in the 18S data set, but their higher internal consistency resulted in a strong phylogenetic signal. Inferred relationships were stable over a wide range of alignment parameters, but the inclusion of alignment-sensitive positions and presumptive gap characters added substantially to the resolution and overall support of the tree. Both data sets were in general congruence. The combined evidence suggests that relationships in Cicindelidae as currently recognized have to be reconsidered. No convincing evidence could be found for the traditional subdivision into two subfamilies, Cicindelinae and Collyrinae. The latter is embedded within the Cicindelinae and clearly not the sister to all other cicindelids. Most of the current tribes and subtribes, particularly in the Megacephalini, are paraphyletic groups, whereas monophyly of the large tribe Cicindelini is well supported by the molecular data.