A previously reported 15-loci STR multiplex was evaluated. Evaluation results indicated that this STR system is not suitable for forensic identification due to identified issues. A novel 13-loci STR multiplex was developed and optimized for cannabis sativa identification (3500 Genetic analyzer), according to ISFG and SWGDAM recommendations, using primer and multiplex STR design software, along with a gradient PCR approach for optimal annealing temperature determination. As a proof of concept, a custom panel for MPS was designed to interrogate 12 cannabis-specific STR loci by sequence rather than by size. A simple workflow was designed to integrate the custom PCR multiplex into a workflow compatible with the Ion Plus Fragment Library Kit, Ion Chef, and Ion SS system. The study resulted in a preliminary investigation of sequence variation for 12 autosomal STR loci in 16 cannabis samples. Results revealed intra=repeat variation in eight loci where the nominal or size-based allele was identical, but variances were discovered by sequence. Although the panel was not fully optimized and only a small number of samples were evaluated, this study demonstrated that more informative STR typing can be performed on a MS platform.