Sperm marking represents a valuable monitoring tool for genetic pest control strategies such as the Sterile Insect Technique, but also provides a key tool for reproductive biology studies. Sperm-marked lines can be generated by introducing transgenes that mediate the expression of fluorescent proteins during spermatogenesis. Homozygous lines established by transgenesis approaches are going through a genetic bottleneck that can lead to reduced fitness. Transgenic SIT approaches have mostly focused on Dipteran and Lepidopteran pests so far. With this study, we provide sperm-marked lines for the Coleopteran pest model organism, the red flour beetle Tribolium castaneum, based on the β2-tubulin promoter/enhancer driving red (DsRed) or green (EGFP) fluorescence. The obtained lines are reasonably competitive and were thus used for studies on reproductive biology confriming the phenomenon of ‘last male sperm precedence’ and that the spermathecae are deployed for long term sperm storage enabling the use of sperm from first matings even after secondary matings for a long period of time. The homozygosity and competiveness of the lines will enable future studies to analyze the controlled process of sperm movement into the long time storage organ as part of a post-mating cryptic female choice mechanism of this extremely promiscuous species.