The aim of this study is to identify and investigate levels of toxic heavy metals in PM2.5 fractioned road dust to better understand the associated inhalation risk and potential health impacts. To achieve this aim, concentrations of seven traffic generated heavy metals (Cu, Pb, Zn, Cd, Ni, Sb, and Cr) were determined in the PM2.5 fraction of road dust samples from four different locations (offices, residential, hospital, and school) in two cities (Karachi and Shikarpur) of Pakistan using ICP-MS. The average concentration values of heavy metals in Karachi were as follows: 332.9 mg/kg Cu, 426.6 mg/kg Pb, 4254.4 mg/kg Zn, 62.3 mg/kg Cd, 389.7 mg/kg Ni, 70.4 mg/kg Sb, 148.1 mg/kg Cr, whereas the average concentration values of heavy metals in Shikarpur were 245.8 mg/kg Cu, 538.4 mg/kg Pb, 8351.0 mg/kg Zn, 57.6 mg/kg Cd, 131.7 mg/kg Ni, 314.5 mg/kg Sb, 346.6 mg/kg Cr. The pollution level was assessed through two pollution indices enrichment factor (EF) and geoaccumulation index (Igeo). These indices showed moderate to extreme level pollution in studied areas of both cities. The health risk assessment through inhalation contact was conducted according to the United States Environmental Protection Agency’s (USEPA) model for children and adults. Both non-cancerous and cancerous risks were characterised in the road dust samples for each location. As yet, there is not a single study on the concentrations of heavy metals in PM2.5 fractions of road dust in Karachi and Shikarpur, findings of this research will facilitate researchers for further investigations in current field.