The effects of dark chilling on CO ₂ assimilation, chlorophyll a fluorescence kinetics and nitrogen fixation were compared in two Glycine max (L.) Merr. genotypes. The aim was to elucidate the mechanisms by which photosynthesis was inhibited as well as identification of selection criteria for dark chilling tolerance. Seedlings were dark chilled (8°C) for 9 consecutive nights but kept at normal day temperatures (28°C). CO ₂ gas exchange analysis indicated that photosynthesis in Maple Arrow was inhibited largely as a result of stomatal limitation, while in Fiskeby V, it indicated inhibition of the mesophyll reactions. Increased intercellular CO ₂ concentration and decreased carboxylation efficiency suggested loss of Rubisco activity in Fiskeby V, although no effect on the K _M (CO ₂ ) of Rubisco was observed. Quantification and deconvolution of the Chl a fluorescence transients into several phenomenological and biophysical parameters (JIP‐test) revealed large genotypic differences in the response of PSII to dark chilling. These parameters differentially changed in the two genotypes during the progression of the chilling treatment. Among them, the performance index, reflecting several responses of the photochemical apparatus, provided the best preliminary overall assessment of the genotypes. In contrast, the quantum yield of primary photochemistry ϕ _Po (F _V /F _M ) was quite insensitive. The recovery of most of the JIP‐test parameters in Maple Arrow after 6 and 9 nights of dark chilling was a major genotypic difference. Genotypic differences were also observed with regard to the ureide response and N ₂ fixation appeared to be more sensitive to dark chilling than CO ₂ assimilation. The JIP‐test provided information consistent with results derived from CO ₂ assimilation and N ₂ fixation studies suggesting that it can substitute the much more time‐consuming methods for the detection of chilling stress and can well satisfy the requirements of a rapid and accurate screening method.