In developing countries, like Pakistan, the pursuit of urbanization and economic development disrupts the delicate ecosystem, resulting in additional biogeochemical emissions of heavy metals into the human habitat and posing significant health risks. The levels of these trace elements in humans remain unknown in areas at higher risk of pollution in Pakistan. In this investigation, selected trace metals including Copper (Cu), Chromium (Cr), Lead (Pb) Cadmium (Cd), Cobalt (Co), Nickel (Ni), and Arsenic (As) were examined in human hair, urine, and nail samples of different age groups from three major cities (Muzaffargarh, Multan, and Vehari) in Punjab province, Pakistan. The results revealed that the mean concentrations (ppm) of Cr (1.1) and Cu (9.1) in hair was highest in Muzaffargarh. In urine samples, the mean concentrations (μg/L) of Co (93), As (79), Cu (69), Cr (56), Ni (49), Cd (45), and Pb (35) were highest in the Multan region, while As (34) and Cr (26) were highest in Vehari. The mean concentrations (ppm) of Ni (9.2), Cr (5.6), and Pb (2.8), in nail samples were highest in Vehari; however, Multan had the highest Cu (28) concentration (ppm). In urine samples, the concentrations of all the studied metals were within permissible limits except for As (34 µg/L) and Cr (26 µg/L) in Vehari. However, in nail samples, the concentrations of Ni in Multan (8.1 ppm), Muzaffargarh (9 ppm), Vehari (9.2 ppm), and Cd (3.69 ppm) in Muzaffargarh exceeded permissible limits. Overall, the concentrations of metals in urine, nail, and hair samples were higher in adults (39–45 age group). Cr, Cu, and Ni revealed significantly higher concentrations of metals in hair and water in Multan, whereas As in water was significantly (p < 0.001) correlated with urinary As in Multan, indicating that the exposure source was region-specific.