Interspecific comparative molecular analyses of transposed genes and their flanking regions can help to elucidate the time, direction, and mechanism of gene transposition. In the Drosophila melanogaster genome, three Larval serum protein 1 (Lsp1) genes (α, β and γ) are present and each of them is located on a different chromosome, suggesting multiple transposition events. We have characterized the molecular organization of Lsp1 genes in D. buzzatii, a species of the Drosophila subgenus and in D. pseudoobscura, a species of the Sophophora subgenus. Our results show that only two Lsp1 genes (β and γ) exist in these two species. The same chromosomal localization and genomic organization, different from that of D. melanogaster, is found in both species for the Lsp1β and Lsp1γ genes. Overall, at least two duplicative and two conservative transpositions are necessary to explain the present chromosomal distribution of Lsp1 genes in the three Drosophila species. Clear evidence for implication of snRNA genes in the transposition of Lsp1β in Drosophila has been found. We suggest that an ectopic exchange between highly similar snRNA sequences was responsible for the transposition of this gene. We have also identified the putative cis-acting regulatory regions of these genes, which seemingly transposed along with the coding sequences.