Theanine, a unique non-protein amino acid, is specifically accumulated in tea plants during winter. This study explored the theanine accumulation patterns in ‘Longjing 43’ and ‘Huangjinya’ under different N supply conditions and analyzed the expression of genes involved in theanine biosynthesis during winter dormancy. We found that the two tea plant cultivars shared similar theanine accumulation patterns in winter. After 30 d of cultivation with various N forms and N deficiency, the theanine content in the tissues of both cultivars was highest in the control group, followed by NH4+ treatment. Furthermore, we noted that root growth of tea plants was inhibited to varying degrees under different N sources and N-deficient conditions. Gene expression analysis revealed that both N forms can induce the transcription of key genes, including CsADC, CsALT, CsCuAO, CsGDH2, CsPAO, CsNiR, CsNR, and CsTS1 in ‘Longjing 43’ and ‘Huangjinya’. The expression of these genes was strongly correlated with theanine levels under the N treatments. The winter theanine accumulation was finely tuned by the interplay of multiple related genes, with expression levels varying across different cultivars and tissues.