Leghaemoglobin content in nodules is closely related to the amount of nitrogen fixed by the legume–rhizobium
symbiosis. It is, therefore, commonly measured in order to assess the effect of growth-promoting parameters such as
fertilisation on the symbiotic nitrogen fixation efficiency of legumes. The cyanmethaemoglobin method is a reference
method in leghaemoglobin content quantification, but this method is time-consuming, requires accurate and careful
technical operations and uses cyanide, a toxic reagent. As a quicker, simpler and non-destructive alternative, a method
based on near infrared (NIR) hyperspectral imaging was tested to quantify leghaemoglobin in dried nodules. Two
approaches were evaluated: (i) the partial least squares (PLS) approach was applied to the full spectrum acquired with
the hyperspectral device and (ii) the potential of multispectral imaging was also tested through the preselection of the most
relevant wavelengths and the building of a multiple linear regression model. The PLS approach was tested on mean
spectra acquired from samples containing several nodules and acquired separately from individual nodules. Peas (Pisum
sativum L.) were cultivated in a greenhouse. The nodules were harvested on four different dates in order to obtain
variations in leghaemoglobin content. The leghaemoglobin content measured with the cyanmethaemoglobin method in fresh
nodules ranged between 1.4 and 4.2 mg leghaemoglobin g–1 fresh nodule. A PLS regression model was calibrated on
leghaemoglobin content measured with the reference method and mean NIR spectra of dried nodules acquired with a
hyperspectral imaging device. On a validation dataset, the PLS model predicted the leghaemoglobin content in nodule
samples well (R2 = 0.90, root mean square error of prediction = 0.26). The multispectral approach showed similar
performance. Applied to individual nodules, the PLS model highlighted a wide variability of leghaemoglobin content in
nodules harvested from the same plant. These results show that NIR hyperspectral imaging could be used as a rapid and
safe method to quantify leghaemoglobin in pea nodules.