Abstract: Enhancing crop tolerance to infertile conditions is crucial for improving the utilization rate of marginal lands. In the current study, 24 foxtail millet samples from three different stages of domestication (wild, landrace, and cultivar) were selected. A hydroponic setup with five low nitrogen concentration gradients was employed 0.05 mmol·L⁻¹ NH₄NO₃ (N1), 0.1 mmol·L⁻¹ NH₄NO₃ (N2), 0.2 mmol·L⁻¹ NH₄NO₃ (N3), 0.4 mmol·L⁻¹ NH₄NO₃ (N4), and 1.0 mmol·L⁻¹ NH₄NO₃ (N5), to compare the comprehensive low-nitrogen tolerance ability among the different genotypes of foxtail millet and to identify efficient physiological indicators for germplasm resource screening. This study aimed to gain a deeper understanding of the tolerance of foxtail millet to low-nitrogen stress at different stages of domestication, providing an important preliminary basis for the exploitation of foxtail millet germplasm resources and genes resistant to low-nitrogen. Our findings showed that all the tested physiological indicators, excluding chlorophyll content of flag leaf and root-to-shoot ratio, from the three domestication stages of foxtail millet showed significant increases in the N3 treatment compared to that in the N2 treatment. However, the plant height, aboveground dry weight, flag leaf area, and underground dry weight of the wild species reached their maximum values at N3 and then stabilized, whereas those indicators from landrace and cultivar species continued to increase. Wild species have a lower demand for nitrogen compared to that in landrace and cultivar species, suggesting that low nitrogen tolerance in foxtail millet has been reduced during domestication and breeding, and the threshold of nitrogen concentration required to maintain normal growth has been increasing. Based on a comprehensive evaluation using the nitrogen tolerance coefficient, it was observed that wild species had better nitrogen tolerance than landrace and cultivar species. Principal Component Analysis (PCA) revealed significant differences in the phenotypes between wild species, landraces, and cultivar species under low nitrogen stress, with aboveground dry weight, underground dry weight, and root volume showing high loadings and accounting value for a significant proportion of the data variance. Thus, the physiological indicators and results derived from the PCA effectively reflect the varying degrees of low-nitrogen tolerance exhibited by foxtail millet across different domestication levels. These results demonstrate that domestication reduces the tolerance of foxtail millet to low nitrogen stress. Aboveground dry weight, because of its convenient detection and close correlation with foxtail millet response to low nitrogen, can be used as a preferred phenotypic indicator for large-scale screening of low-nitrogen-tolerant foxtail millet varieties in the future. Our study provides a reference for efficient screening of foxtail millet varieties under infertile conditions.