Marijuana is a plant cultivated and trafficked worldwide as a source of fiber (hemp), medicine, and intoxicant. In the current project, the STR multiplex consisted of 13 previously described C. sativa STR loci: ANUCS501, 9269, 4010, 5159, ANUCS305, 9043, BG05, 1528, 3735, CS1, D02, C11, and H06. A sequenced allelic ladder that consisted of 56 alleles was designed to accurately genotype 101 C. sativa samples from three seizures provided by a U.S. Customs and Border Protection crime lab. Using an optimal range of DNA (0.5-1.0 ng), validation studies revealed well-balanced electropherograms (inter-locus balance range: 0.500-1.206), relatively balanced heterozygous peaks (mean peak height ratio of 0.83 across all loci) with minimal artifacts and stutter ratio (mean stutter of 0.021 across all loci). This multi-locus system is relatively sensitive (0.13 ng of template DNA) with a combined power of discrimination of 1 in 55 million. The 13 STR panel was found to be species specific for C. sativa; however, non-specific peaks were produced with Humulus lupulus. The results of this research show the robustness and applicability of this 13 loci STR system for forensic DNA profiling of marijuana samples (Publisher abstract modified)