Introduction: Simple questionnaires based on so called "neuropathic pain descriptors" are the most frequently used screening tools in the diagnosis of neuropathic pain, especially by non-specialists. However, no validated Czech version of any of the internationally accepted nueropathic pain questionnaires is available. Aim: The aim of this study was to develop and validate a Czech version of the Neuropathic Pain Symptom Inventory (NPSI), a questionnaire that allows the user to characterize subtypes of neuropathic pain, is sensitive to change, and has discriminatory power. Patients and methods: The NPSI was translated into the NPSIcz using the forward-backward translation method and validated in a group of patients suffering from neuropathic pain of peripheral (n = 66) or central origin (n = 30) due to polyneuropathy or multiple sclerosis, and in a group of individuals suffering from nociceptive pain (due to severe osteoarthritis of the knee or hip joint; n = 70). The NPSIC2 was repeated within three hours in 19 individuals to determine test-retest reliability. Results: The NPSICZ showed good diagnostic validity in discriminating between patients with neuropathic and nociceptive pain (p < 0.001) but low ability to discriminate between patients with central and peripheral neuropathic pain (p > 0.05). Repeated administration confirmed acceptable test-retest reliability (intra-class correlation coefficient of 0.86 for the NPSIC2 sum score). Cluster analysis identified six different pain profiles. Conclusions: The NPSICZ is able to discriminate between neuropathic and nociceptive pain, to identify different pain profiles, and has acceptable test-retest reliability. It can, therefore, be recommended as an easy-to-administer questionnaire suitable for the diagnosis of neuropathic pain, including long-term follow up.