We have developed a novel STR 25-plex florescence multiplex-STR kit (DNATyper25) to genotype 23 autosomal and two sex-linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non-CODIS. The sex-linked markers include a Y-STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non-human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration <= 60 ng/mu L and hematin concentration <400 mu M. For forensic evaluation, the 23 autosomal STRs followed the Hardy-Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 x 10(-22), TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.