The identification of the intersegmental plane isa major interoperative challenges during pulmonary segmentectomies. The objective of this pilot study is to test the feasibility of lung perfusion assessment by Hyperspectral Imaging for identification of the intersegmental plane. A pilot study (clinicaltrials.org: NCT04784884) was conducted in patients with lung cancer. Measuring tissue oxygenation (StO2; upper tissue perfusion), organ hemoglobin index (OHI), near-infrared index (NIR; deeper tissue perfusion) and tissue water index (TWI), the Hyperspectral Imaging measurements were carried out in inflated (Pvent) and deflated pulmonary lobes (PnV) as well as in deflated pulmonary lobes with divided circulation (PnVC) before dissection of the lobar bronchus. A total of 341 measuring points were evaluated during pulmonary lobectomies. Pulmonary lobes showed a reduced StO2 (Pvent: 84.56%±3.92 vs. PnV: 63.62%±11.62 vs. PnVC: 39.20%±23.57; p<0.05) and NIR-perfusion (Pvent: 50.55±5.62 vs. PnV: 47.55±3.38 vs. PnVC: 27.60±9.33; p<0.05). There were no differences of OHI and TWI between the three groups. This pilot study demonstrates that HSI enables differentiation between different ventilated and perfused pulmonary tissue as a precondition for HSI segment mapping.