Analysis of short tandem repeats (STRs) using capillary electrophoresis (CE) is the current standard for forensic DNA analysis. However, massively parallel sequencing (MPS) of STRs has the potential to provide greater discriminating power through the use of sequence-defined alleles. By determining the base pair sequence of an STR, alleles of the same length may be differentiated. The ForenSeqTM MainstAY Kit from Verogen is an MPS kit designed to analyze forensically relevant samples on the Illumina MiSeq FGxTM System. To assess the ForenSeqTM MainstAY Kit’s ability to analyze evidence found in forensic casework, samples were selected from a set of differential extraction samples from a previous study. The samples had been extracted using either the Qiagen EZ1® DNA Investigator® Kit or a Temperature Controlled Differential Extraction (TCDE) method and analyzed using CE. The selected samples were prepared with the ForenSeqTM MainstAY Kit in 2 replicates, along with a dilution series to assess the sensitivity of the kit. The results showed that the ForenSeqTM MainstAY Kit was able to produce profiles of similar quality for forensic comparison to CE. Although read counts were lower than expected at some concentrations, at least one of the dilution series replicates produced a full profile at concentrations as low as 0.4 ng. Total read count per locus varied, with the PentaD and PentaE loci producing the lowest mean read counts. Heterozygote balance between sequencing replicates was consistent. However, CE did display better heterozygote balance than MPS. Most importantly, isoalleles were detected at the D21S11, D8S1179, D9S1122, D3S1358, and D13S317 loci and could be used to determine the genotypes at a locus with two contributors. Overall, the ForenSeqTM MainstAY Kit could be a valuable asset to the field of forensic DNA analysis for its ability to distinguish alleles by sequence as well as by length.