This study investigated the cold tolerance of a laboratory-reared population and latitude-separated populations of the leafminer Liriomyza sativae in China in terms of low-temperature survival rate and supercooling capacity. Pupae of the laboratory-reared population are susceptible to freezing temperatures. The supercooling point of the pupae varied at a maximum of up to 10°C among the geographic populations. Both acclimation of the pupae at 5 and 10°C significantly increased the cold survival rate of laboratory-reared and field-collected populations. But the field population had a more active response to the same acclimation regime. Combining the experimental data with previous field investigations, the −2°C isotherm of the minimum mean temperature of January was proposed as the leafminer's over-wintering range limit. Meanwhile, with the widespread availability of greenhouses as a source of re-infestation in northern China, the leafminer's natural border of distribution should be determined by the minimum temperature in warm seasons. Our results suggest that biologically similar Liriomyza species may cope with the intensifying cold stress along the latitude by adopting a mixed cold-tolerance strategy, which is closely associated with the greenhouse microhabitats. The physiologically based modeling of the over-wintering limit provides a tool for guiding the management for greenhouse pests and predicting the source of pest infestation.