ABSTRACT: In this study, a new STRtyper-27 system, including the same Yfiler Plus loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS449, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627 and DYF387S1a/b), was established using a set of 5 fluorescent dye labels. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house for this multiplex system. This paper describes the validation studies conducted with the STRtyper-27Y system using a 3130XL genetic analyzer for fragment length detection that included the analysis of the following parameters and aspects: sensitivity, species specificity, inhibition, haplotype concordance, precision, stutter, DNA mixture