The use of treatment wetlands (TWs) presents particular challenges in regions with sub-zero winter temperatures, due to reduced biological activity and risk of pipe breakage or clogging due to freezing. We studied the vertical temperature distribution in four pilot-scale TWs exposed to winter temperatures in order to determine the impact of operational system parameters and the role of insulation on heat conservation inside the filtering bed. The overall temperature pattern was similar in all wetlands, with a trend of increasing temperature from the surface toward the bottom during the cold season. No freezing was detected in the wetlands despite average daily temperatures as low as −20 °C. Influent water temperature and hydraulic loading had a stronger influence on TW temperatures in winter than air temperature. The vertical distribution of temperatures in TWs is more sensitive to hydraulic loading variation in the percolating operating condition than in the saturated flow with forced aeration configuration. Our results suggest that TW systems can remain operational under cold winter conditions provided the surface is properly insulated by vegetation, mulch and/or snow.