Defining temperature at depth to identify geothermal resources relies on the evaluation of the Earth heat flow based on equilibrium temperature measurements as well as thermal conductivity and heat generation rate assessment. Such high-quality geothermal data can be sparse over the region of interest. This is the case of the St. Lawrence Lowlands sedimentary basin covering 20,000 km2 to the south of Quebec, Canada, and enclosing only three wells up to a depth of 500 m with equilibrium heat flow measurements. However, more than 250 oil and gas exploration wells have been drilled in this area, providing for this study (parce que c'est 93 sinon) 81 locations with bottom-hole temperature up to a depth of 4300 m, however, not at equilibrium. Analyzing these data with respect to the deep geothermal resource potential of this sedimentary basin requires evaluating the thermal conductivity and heat generation rate of its geological units to properly extrapolate temperature downward. This was done by compiling literature and recent thermal conductivity measurements in outcrop and core samples as well as new heat generation rate estimates from spectral gamma ray logs to establish a first thermal assessment of geological units deep down into the basin. The mean thermal conductivity of the thermal units varies from 2.5 to 6.3 W/m·K, with peak values in the basal sandstones, while the heat generation rate varies from 1.6 to 0.3 µW/m3, decreasing from the upper caprocks toward the base of the sequence. After correcting the bottom-hole temperatures for drilling disturbance with the Harrison correction and subsequently for paleoclimate variations, results indicate a mean geothermal gradient of 23.1 °C/km, varying from 14 to 40 °C/km. Evaluating the basin thermal state from oil and gas data is a significant challenge facilitated by an understanding of its thermal properties.