The tri-allelic pattern is a genotyping abnormality that can be observed during routine short tandem repeat (STR) profiling in the field of forensic science. Fourteen tri-allelic patterns have been observed in nine different loci during routine forensic casework consisting of 20,000 STR profiles. All these 20,000 STR unrelated profiles were profiled using AmpFlSTR Identifiler Plus® and Global Filer® kits. Tri-allelic patterns can be divided into two types based on RFUs (relative fluorescent units) in peaks of three component alleles. Unequal RFUs of all three peaks are observed in the Type-I pattern, whereas in the Type-II pattern, RFUs of all three peaks are nearly equal. A total of nine novel tri-allelic genotypes were observed out of 20,000 unrelated individuals in the Pakistani population. All of these forensic cases belonged to the Type-I pattern which means that the sum of the height of two smaller peaks is nearly equal to the height of the third larger peak. The frequency of occurrence for all these patterns was compared with already reported data. In this study, eight novel tri-allelic patterns have been reported which are not listed in the National Institute of Standards and Technology (NIST) database as well as in any published article.