Three geochemically and stratigraphically distinct oil families are found in southwestern Ontario; the Cambro-Ordovician, Silurian and Devonian families. In this study, a total of 34 crude oil samples representing all the major producing horizons were investigated by means of open column liquid chromatography, gas chromatography (GC) and gas chromatography-mass spectrometry (GCMS) techniques. The separation into three oil families corresponds well to variability in gross composition and the gasoline range characteristics. The degree of paraffinicity suggests that the Devonian oils are of the lowest and the Cambro-Ordovician of the highest thermal maturity. Cambro-Ordovician and Devonian families show some affinities in their biomarker signatures such as similar normal alkane distributions, low amounts of pristane (Pr) and phytane (Ph) relative to n-alkanes, almost identical Pr/Ph ratios, predominance of Ts over Tm, low gammacerane concentrations and abundant C-29 and C-27 diasteranes. However, different Carbon Preference Index (C-14-C-20 range), middle-/long-chain n-alkanes, hopanes/steranes and diasteranes/regular steranes ratios as well as the presence of C-30 4-desmethylsteranes in the Devonian oils (absent in Ordovician oils) clearly separate both families. The Silurian family has a distinctly different geochemical composition characterized by a broad, bimodal n-alkane distribution with maxima at C-15 and C-23 to C-25, high concentrations of acyclic isoprenoids with Ph dominating over Pr, a predominance of Tm over Ts, a high concentration of gammacerane, abundant homohopanes with prominent C-34 member, low concentrations of tricyclic terpane relative to the C-24 tetracyclic terpane and a relatively low abundance of rearranged steranes. The triaromatic steroids ratios (TA I/TA[I+II]) and isomerization of C29 steranes (S/[S+R] and beta beta/[alpha alpha+beta beta]) indicate that maturity increases from Devonian through Silurian to Ordovician oils. Overall, biomarker characteristics indicate a carbonate/evaporite source rock deposited under hypersaline conditions in a strongly reducing environment for the Silurian oils, and a elastic source formed under dysoxic to anoxic conditions for the Devonian and Cambro-Ordovician oils. Subtle compositional irregularities of Devonian oils are attributed to geographical variabilities in the maturity and composition of source kerogen. Depending on their geographical proximity, Devonian oils may be genetically related to source rocks deposited in either the Michigan or Appalachian basins, or both. Geochemical variations amongst oils of the Silurian family not only identify at least two subfamilies, probably related to variations in their source rocks, but also indicate localized sources and short secondary migration pathways. The geochemical character of the Cambro-Ordovician oil family is typical for oils derived from Ordovician-a,oed source rocks. However, there is some evidence for a separate source for Cambrian oils. Moreover, variability in gasoline and biomarker parameters indicate that Ordovician oils were generated from different source rocks located in the stratigraphic intervals of their reservoirs (Black River and Trenton, respectively).