Study results show that Pyrosequenching is a useful tool for typing Y chromosomal STRs, and it can provide a complement to conventional forensic Y STR analyses; moreover, it can be used to rapidly evaluate novel markers. At four loci, (DYS389 II, DYS390, DYS391, and DYS393), the Pyrosequencing analysis revealed sequence variants in frequencies between 1.43 percent and 14.3 percent. Using 8 markers, 57 unique haplotypes were observed with a discrimination capacity of 0.81. Pyrosequencing is a non-electrophoretic DNA sequencing technique based on detection of pyrophosphate during DNA synthesis. It was first developed for SNP analysis or sequencing of short stretches of DNA. It has been used in mitochondrial analysis for forensic identification. In the current research, a Pyrosequencing-based Y-STR analysis assay was developed for the markers DYS19, DYS389 I/II, DYS390, DYS391, DYS392, DYS393, and DYS438. One of the major advantages of the Pyrosequencing technique is its capacity to observe sequence variants. Variants with a constant size (i.e., SNPs) cannot be detected with standard fragment length analysis and allelic ladders. Seventy unrelated Swedish men were typed for the 8 Y chromosomal markers. The descriptions of material and methods address the DNA samples, primer design and PCR, and template preparation and sequence analysis. 3 tables, 4 figures, and 37 references