Background: Patients suffering from chronic spontaneous urticaria (CSU) are typically classified as type I or type IIb autoimmune CSU, but further patient stratification is hindered by the lack of biomarkers. Objectives: We investigated whether the histamine content of individual basophils differ between patient subtypes in CSU to evaluate its potential as a biomarker. Methods: A total of 101 patients diagnosed with CSU were included in the study. The histamine content per circulating basophil was derived from the basophil count in peripheral blood and levels of total cellular blood histamine. These measures, together with results from the serum-induced basophil histamine release assay (s-BHRA), were correlated to information on demographics, clinical characteristics, patient reported outcomes and laboratory analyses. Results: The histamine content per basophil was significantly different between s-BHRA negative and -positive patients (0.175 vs. 1.40 pg/cell, p < 0.001) and showed a significant negative correlation to the degree of basophil activation in s-BHRA (ρ = −0.209, p = 0.036). Furthermore, the amount of histamine in individual basophils was found to be significantly correlated with levels of total cellular blood histamine (ρ = 0.376, p < 0.001), eosinophil counts (ρ = 0.205, p = 0.040), levels of thyroid stimulating hormone (ρ = −0.246, p = 0.014) and titre of antibodies against thyroid peroxidase (ρ = −0.216, p = 0.031) and thyroglobulin (ρ = −0.203, p = 0.044). Conclusions: Low content of intracellular histamine in circulating basophils is associated with known markers of type IIb autoimmune CSU. Further studies are required to assess whether the amount of histamine in basophils can be used to monitor or predict response to treatment in patients.