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Forensic analysis of human identification gender must be considered important for the testing of mixed male and female samples. In our experiment, the amelogenin gene is used for the investigation of human gender and the HirisPlex tool for eye colour prediction. A single primer pair was used to amplify the amelogenin gene. The effectiveness of the primer has been tested in known humans. For the PCR band pattern, the X-linked gene partially produced a 106 -bp fragment and the Y-linked gene produced a 112 -bp fragment. All human test results showed a male sample with two bands (106 -bp and 112 -bp) and a female sample with only one band (106 bp). We also find out the eye colour of individuals after gender confirmation through an online eye colour prediction tool. The HirisPlex tool accurately predicted the blue and brown eyes of Pakhtoon population living in Malakand division. These findings are reliable techniques for tracking the AMELX/Y gene as a marker for polymerase chain reaction testing for gender identity. It also suggests that the HIrisPlex tool is ideal for the prediction of blue and brown eye colours.