Objective: To assess the efficacy of a mini-short tandem repeat (mini-STR)-based next-generation sequencing (NGS) method for non-invasive prenatal paternity testing (NIPPT). Design: Methodological study. Setting and population: NIPPT was performed for 28 families, which consisted of pregnant women, their husbands, and infants. Methods: Plasma DNA from 28 pregnant women was extracted and cell-free foetal DNA (cffDNA) genotyping was performed at 23 mini-STR loci using the Illumina NextSeq 500 system. For each mini-STR locus, the cffDNA genotype was validated by determination of infant DNA genotype. The mini-STR loci with high accordance rates were selected for comparison of STR genotyping results between cffDNA and biological father DNA or random male DNA for each family. Main outcome measures: The mini-STR-based NGS method can be used for NIPPT. Results: The biological relationship was validated between alleged fathers and infants in all 28 families using the capillary electrophoresis (CE) method. Moreover, the accordance rates of STR genotypes D5S818, D19S253, and D21S1270 were less than 50% in 23 autosomal STR loci. The STR genotype matching probability was calculated using 20 STR loci with more than 60% accordance rate. There was a significant difference in the STR genotype matching probability between cffDNA and the DNA from the biological father (75–100%) or from random males (25–70%) (P < 0.0001). Conclusions: Our study demonstrated that mini-STR can be used for NGS-based NIPPT. Furthermore, this method can be used for crime control purposes using the STR data available from the national forensic DNA databases.