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DNA profiling--inclusive sex determination-- with microsatellite markers is currently a commonly used genetic method of studying humans. An efficient technique of producing the genetic data is amplification of multiple microsatellites in a single PCR reaction. Here we introduce a novel PCR-multiplex system for analysis of four polymorphic Y-STRs. Specifically, these are DYS449, DYS456, DYS458, and DYS464. These loci were chosen because of their reported high diversity in Euroamerican population (10), as well as their absence in the commercial analytical kits at the time of beginning of this study. Our objective was to design this PCR-multiplex for use of fragmentation analysis by electrophoresing samples on a capillary semi-automated genetic analyzer applying only one fluorescent dye. The PCR system we propose, may be notably used in fields such as forensic and human population genetics.