AbstractMiscanthusis a C4perennial grass being developed for bioenergy production in temperate regions where chilling events are common. To evaluate chilling effects onMiscanthus,we assessed the processes controlling net CO2assimilation rate (A) inMiscanthus x giganteus(M161) and a chilling‐sensitiveMiscanthushybrid (M115) before and after a chilling treatment of 12/5 °C. The temperature response ofAand maximum Rubisco activityin vitrowere identical below 20 °C in chilled and unchilled M161, demonstrating Rubisco capacity limits or co‐limitsAat cooler temperatures. By contrast,Ain M115 decreased at all measurement temperatures after growth at 12/5 °C. Rubisco activityin vitrodeclined in proportion to the reduction inAin chilled M115 plants, indicating Rubisco capacity is responsible in part for the decline inA. Pyruvate orthophosphate dikinase activities were also reduced by the chilling treatment when assayed at 28 °C, indicating this enzyme may also contribute to the reduction inAin M115. The maximum extractable activities of PEPCase and NADP‐ME remained largely unchanged after chilling. The carboxylation efficiency of the C4cycle was depressed in both genotypes to a similar extent after chilling. ΦP:ΦCO2remained unchanged in both genotypes indicating the C3and C4cycles decline equivalently upon chilling.