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As global temperature rise, the threat of heat stress to rapeseed production is becoming more obvious. Exploring the response characteristics of two important biological pathways, oil accumulation and photosynthesis, to heat stress during B. napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed. The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B. napus germplasms with different oil content and environmental sensitivity, including 6 rapeseed varieties which exhibited environmentsensitive/insensitive and with high, medium or low oil content, were tested by whole plant heat stress or the in vitro silique culture system. Both assay exhibited similar trend on oil content of the rapeseed germplasms. The heat effect on the chlorophyll fluorescence kinetic parameters Fv/Fm, ETR and Y(II) were also consistent. Heat stress significantly decreased oil content, although there was abundant genetic variation on heat tolerance among the genotypes. Correlation analysis showed that the decrease rate of Fv/Fm of silique heat-stressed B. napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed (R = 0.9214, P-value < 0.01). Overall, the results indicated that heat stress inhibited oil accumulation and photosynthesis in B. napus developing seed. The decrease rate of chlorophyll fluorescence parameter Fv/Fm of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification. Further, two heat insensitive rapeseed varieties with high oil content were identified. [ABSTRACT FROM AUTHOR]