In this study, a novel 27-locus system (now known as the SureID PanGlobal system), including 24 autosomal STRs (D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D2S441, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33, D10S1248), two Y-chromosome markers (DYS391 and Y-indel) and the sex determining marker, Amelogenin was developed with six fluorescent dyes labeling. The included STR loci belonged to the core loci in the Combined DNA Index System (CODIS) and the European Standard Set (ESS) as well as some additional loci commonly used in commercial kits and national DNA databases. This paper describes the validation studies conducted with the SureID PanGlobal system using Applied Biosystems 3500 XL Genetic Analyzer for fragment length detection that included the analysis of the following parameters and aspects: PCR conditions, sensitivity, species specificity, inhibition, precision, stutter, DNA mixtures, and stability studies with crime scene samples. The studies demonstrated that the SureID PanGlobal system is reproducible, accurate, sensitive and robust for forensic application and databasing. Additionally, the whole cycling time of the system can finish within 65 minutes, which was developed specifically for rapid and reliable generation of DNA profiles obtained from blood, buccal swabs and forensic stains.