Numerous sequencing studies, including transcriptomics of host-pathogen systems, sequencing of hybrid genomes, xenografts, mixed species systems, metagenomics, and metatranscriptomics, involve samples containing genetic material from divergent organisms. A crucial step in these studies is identifying from which organism each sequencing read originated, and the experimental design should be directed to minimize biases caused by cross-mapping of reads to incorrect source genomes. Additionally, pooling of sufficiently different genetic material into a single sequencing library could significantly reduce experimental costs but requires careful planning and assessment of the impact of cross-mapping. Having these applications in mind we designed Crossmapper, the first to our knowledge tool able to assess cross-mapping prior to sequencing, therefore allowing optimization of experimental design.