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In an attempt to significantly increase the discriminatory potential of Y-STR systems available to the forensic community, two Y-STR multiplex systems have been developed and validated. The two systems were developed to supplement the recommended nine "minimal haplotype loci," used extensively throughout the European forensic community. In accordance with multiplex development requirements, attempts were made to construct highly discriminatory multiplex systems with an increased number of Y-STR loci contained therein, while seeking to ensure a high degree of sensitivity (1-3 ng of template DNA) and specificity (no artifact production due to confounding female DNA). Two Y-STR multiplex systems, Multiplex ill (MPIII) and Multiplex IV (MPIV), have been developed. They allow for the co-amplification of nine and twenty-one novel Y-STR loci, respectively. Both multiplexes performed well over a wide range of primer, magnesium, and DNA polymerase concentrations. A number of performance checks were employed to characterize the system including sensitivity, specificity, discriminatory capacity and non-probative casework studies. Full male profiles can be obtained with as little as 50 pg of te􀀫plate DNA with Multiplex ill and Multiplex IV, and both systems exhibited a lack of significant female DNA derived artifacts when tested with female DNA alone or in a male/female DNA admixture. Cervicovaginal samples taken up to 48 hours after intercourse resulted in full haplotype determination with both multiplexes. These results demonstrate the potential usefulness of Multiplex III and Multiplex IV for forensic casework.