The temperature field variation law and distribution characteristics of an orthotropic flat steel box girder under sunny conditions were analyzed through a field temperature test on the steel box girder of the operational Runyang Yangtze River Bridge (the suspension bridge part). Function optimization fitting and error analysis of the test data were conducted. A temperature gradient distribution curve applicable to a hexagonal flat steel box girder was proposed. Based on the measurement results, the temperature effect of an orthotropic flat steel box girder was analyzed using finite element method and the effects of different temperature gradient modes on the mechanical characteristics and stress distribution of the steel box girder were compared. Under sunny conditions, heat conduction in the flat steel box girder structure shows distinct "box-room effect" characteristics, and the actual temperature gradient distribution is inconsistent with the one suggested by the existing standards. The thermal stress of a steel box girder calculated from the measured temperature gradient mode exceeds that calculated from the standard, and the intensity approximates that under the action of designed vehicle loads. The temperature-induced stress is distributed centrally near the manufacturing welds of the orthotropic steel box girder, which should be considered in design, construction and research. Results from this study could supplement the existing bridge and culvert design standards.