Here, we present for the first time, a method to generate homozygous segmental introgressions, by means of crosses between a pair of synmorphic species. The introgressions were monitored by the cytogenetic method of polygenic chromosome asynapses. Later the introgressions were evaluated in their capacity to produce sterility in segmental males. Also, the smallest segment with the capacity to produce sterility in segmental males was mapped by in situ hybridization of polythene chromosomes, using 8 sequences of BACs clones as probes. Finally, a bioinformatic analysis was carried out to identify the presence of particular genes. From 2 parental strains, D. buzzatii and D. koepferae, 6 simple segmental hybrid lines were generated, whose introgressing segments are distributed along chromosome 4 of these species. From the 6 simple segmental lines and by means of a new crossing strategy, the 6 respective homozygous segmental hybrid offspring were obtained, each of them carrying a specific homozygous introgression. None of the 6 heterozygous introgressions was capable of producing sterility in segmental males, while 4 of the same homozygous introgressions produced total sterility in segmental males, including in this group the two smallest introgressive segments, one of 5.03 % and the other 7.87% with respect to the total length of chromosome 4, which are located in the region F2 to F4 of the standard cytological map based on polythene chromosomes of the Drosophila Repleta group. In situ hybridization, using 8 clones from contig 1065 located along the F2 to F4 region of the physical map of D. buzzattii constructed in BACs, confirmed the precise location of the 6 clones in the chromosomal region F2 to F4 of chromosome 4 of the polygenic chromosomes of both D. buzzatii and D. mojavensis. The bioinformatic analysis of the F2 to F4 region, using the complete genetic sequence of the contig 1065 of D. buzzatti shows the presence of two predicted genes in the genomic map of D. buzzatii (g.1313.t1 and g.1314.t1), and the orthologous association of these 2 genes both with the D. moj_GI22766 gene of D. mojavensis and with the Trivet gene of D. melanogaster.