With global warming, the impact of high-temperature stress on crop production is one of the major issues facing agriculture. Combining a series of field and controlled environment experiments, the current study aimed at understanding causes of yield loss due to heat stress in lima bean (Phaseolus lunatus L.) in order to guide breeding for heat tolerance. A growth-stage analysis of multi-year field trials suggested that yields were reduced by sensitivity to heat during floral transition. High nighttime temperatures during this period were also associated with delayed pod set and subsequent harvest. These effects were validated in controlled environments, with additional tests revealing how nighttime heat stress generally reduces seeds per pod but can also reduce the number of pods set. Using an intra-plant dual temperature treatment design for vegetative and reproductive organs, it was determined that effects on pollen viability and release are primary factors underlying heat stress-associated yield losses in lima bean. Elevated nighttime temperatures did not reduce aboveground biomass, indicating the physiological basis for temperature sensitivity was not driven by a photosynthate deficit, but instead could be attributed to changes in source-sink dynamics. Still, the basis for sensitivity to heat differed among genotypes, opening new targets for a multi-mechanistic breeding approach for heat tolerance. Seed yield in lima bean is reduced when heat stress occurs during reproductive phase.Sensitivity to heat stress is greater during the night.High nighttime temperatures affect multiple physiological mechanisms of reproduction.Genotypes show mechanism-specific sensitivities to high nighttime temperature.Multiple physiological mechanisms may be targeted for breeding heat tolerance in lima bean.