SIGNIFICANCE: Conventionally, spectral photoacoustic imaging (sPAI) to assess tissue oxygenation ( AIM: We demonstrate the second near-infrared (NIR-II) tissue optical window (950 to 1400 nm) for the assessment of blood and tissue APPROACH: The NIR-II PA spectra of oxygenated and deoxygenated hemoglobin were first characterized using a phantom. Optimal wavelengths to minimize spectral coloring were identified. The resulting NIR-II PA imaging methods were then validated RESULTS: sPAI of whole blood, in a phantom, and of blood in kidneys CONCLUSIONS: sPAI using NIR-II wavelengths improved the accuracy of tissue