Real-world driving conditions are essential for measuring pollutant emissions and fuel consumption of vehicles. Real driving emission measurement was recently required for heavy-duty vehicles, previously tested only on the engine dynamometer over standardized operating conditions. This paper describes the results of an experimental campaign carried out on three different EURO VI urban buses fuelled by natural gas, aimed at measurement of carbon monoxide (CO), nitrogen oxides (NOx), carbon dioxide (CO2), particulate matter (PM) emissions and fuel consumption (FC) in real driving conditions. Buses, used for public transport in 3 different Italian cities, were equipped with a portable emission measurement system (PEMS) and tested over line routes, representative of small, medium, and large urban areas. The actual driving cycles were analyzed to understand the differences in exhaust emissions and fuel consumption between the vehicles. Cold start conditions were also investigated. The results showed that the main differences in pollutant emissions between tested buses can be attributed to the calibration of the fuel supply system and to the driving cycle characteristics. Cold-start strongly impacted CO and NOx emissions because of the effect of temperature on catalytic converter efficiency, with cold over-emissions strongly dependent on the ambient temperature at engine start.