With its rapidly growing population and large industrial base, mega city Karachi, Pakistan has been subjected to an increasing amount of ambient particulate matter (PM). The 24-hour fine particulate matter (PM2.5) in Karachi was collected from January 8-January 29, 2006. Samples were extracted and analyzed by gas chromatography mass spectrometry (GC-MS) and ultra-performance liquid chromatography (UPLC) coupled to a triple quadrupole mass spectrometric detector (TQD) and time-of-flight (TOF) mass spectrometer. Daily PM2.5 levels ranged from 99.5 to 251.1 μg m-3 with a mean of 177.4 μg m-3, averaging 4–10- fold higher than the WHO guideline for 24-hour averaged PM2.5 (25 µg m-3). We found that day-of-the-week variation of PM2.5 demonstrate Sundays have significantly lower concentrations (t-test significant at the 95% confidence interval), indicating that weekly behavioral patterns affect local PM2.5 concentrations. A significant negative correlation was found between the daily concentrations of levoglucosan, a biomass burning tracer, and daily average temperatures (r = -0.589, p = 0.004), implicating heating as a major source of biomass burning emissions. Results indicated that polyaromatic hydrocarbons, hopanes, steranes, and alkanes are mainly emitted from fossil fuels and combustion of carbonaceous-containing materials. Organosulfates (OS) and sulfonates were also quantified; significant correlations between OS and sulfonates indicate origination from a common source and similar formation mechanisms, while correlations with hopanes, steranes, and levoglucosan indicate their emissions from primary sources such as fossil fuel and biomass burning. Qualitative analysis suggests the presence of a C6-C9 alkyl sulfate series. Through this study, the chemical composition and source origination of the organic fraction of PM2.5 in Karachi is evaluated for the first time and indicates a strong anthropogenic influence on combustion emissions, particularly biomass and fossil fuel burning.