Sub‐optimal night temperatures below 15°C (dark chilling) frequently reduce soybean [Glycine max (L.) Merrill] production. Nitrate application is known to alleviate some of the negative effects of low root zone temperatures, probably by counteracting the inhibition caused by decreased symbiotic nitrogen fixation (SNF). Under field conditions, however, dark chilling is frequently not accompanied by low root zone temperatures. The possibility that nitrate might increase dark‐chilling tolerance under these conditions is still largely unexplored. In addition to quantifying vegetative development by means of the plastochron index, O–J–I–P (O–I₁–I₂–P) chlorophyll α fluorescence transients were recorded in soybean genotypes of contrasting chilling tolerance during and following exposure to dark chilling in the absence of low root zone temperatures. Plants, inoculated with the N₂‐fixing bacteria, Bradyrhizobium japonicum , were grown with and without nitrate supplementation. The recorded O–J–I–P chlorophyll α fluorescence transients were analysed by the so‐called JIP‐test which translates stress‐induced alterations in these transients to changes in biophysical parameters that quantifies the energy flow through photosystem II (PSII). One of these parameters, the performance index (PI_ABS ), combines the three main functional steps (light energy absorption, excitation energy trapping, and conversion of excitation energy to electron transport) of photosynthetic activity by a PSII reaction centre complex into a single multiparametric expression. By using the PI_ABS we could convincingly show that nitrate supplementation considerably enhances dark‐chilling tolerance and recovery capacity of plants in the absence of low root zone temperatures. This was especially true for the chilling‐sensitive genotype (‘Java 29’), suggesting that the response of SNF to dark chilling might be an important factor contributing towards genotypic differences in chilling tolerance. Our results corroborated previous reports about the superior chilling tolerance of ‘Maple Arrow’, a chilling‐tolerant genotype. The results obtained indicated that the PI_ABS is a far more sensitive indicator of dark‐chilling stress than the maximum quantum yield of primary photochemistry (F_V/F_M).