This thesis seeks to estimate CO2 emissions on a portion of the U.S. 101 highway in San Luis Obispo County before and after construction of a truck only lane on the Cuesta Grade. Towards that aim, the microsimulation software, VISSIM, was used in conjunction with the Environmental Protection Agency’s emissions model, MOVES. The microsimulation model was calibrated and validated against historical and present traffic volumes obtained from Caltrans with good results using several validation measures. It was found that CO2 emissions did decrease between 1998 and 2012 (pre and post lane addition), but this effect was shown to be different for the northbound (uphill) and southbound (downhill) directions. It was shown that the truck lane in the northbound (uphill) direction had a 9.5% decrease in volume with 10.7% decrease in emissions, and the southbound (downhill) direction had a 20.3% increase in volume but 7.4% decrease in emissions. For the northbound (uphill) direction, emissions seemed to correlate more closely with volumes, while the southbound (downhill) direction was less sensitive to these changes.