STRs vary not only in the length of the repeat units and the number of repeats but also in the region with which they conform to an incremental repeat pattern. Massively parallel sequencing (MPS) offers new possibilities in the analysis of STRs since they can simultaneously sequence multiple targets in a single reaction and capture potential internal sequence variations. Here, we sequenced 34 STRs applied in the forensic community of China with a custom-designed panel. MPS performance were evaluated from sequencing reads analysis, concordance study and sensitivity testing. High coverage sequencing data were obtained to determine the constitute ratios and heterozygous balance. No actual inconsistent genotypes were observed between capillary electrophoresis (CE) and MPS, demonstrating the reliability of the panel and the MPS technology. With the sequencing data from the 200 investigated individuals, 346 and 418 alleles were obtained via CE and MPS technologies at the 34 STRs, indicating MPS technology provides higher discrimination than CE detection. The whole study demonstrated that STR genotyping with the custom panel and MPS technology has the potential not only to reveal length and sequence variations but also to satisfy the demands of high throughput and high multiplexing with acceptable sensitivity.