Objective To investigate the prevalence and intensity of pain perception during diagnostic hysteroscopy in women and potential related factors.Methods A total of 489 women were investigated at an infertility clinic. Fluid diagnostic hysteroscopy was performed without analgesia or anesthesia by gynecologists with different levels of experience in operative hysteroscopy, using a 2.9mm rigid scope. The Visual Analog Scale was used to score pain intensity after vaginal speculum insertion and after hysteroscopy. Data collected included age, ethnicity, body mass index, history of infertility and endometrial surgery (curettage and/or hysteroscopy), smoking habits, and hysteroscopy diagnosis. Only the state of anxiety was assessed by the State-Trait Anxiety Inventory given to each patient before the procedure.Results Hysteroscopy median (25th to 75th) Visual Analog Scale scored 3.3 (3 to 5), and 41.7% of the women referred Visual Analog Scale score ≥4. Median (25th to 75th) State-Trait Anxiety Inventory score was 42 (38 to 45), and 58.3% of the women referred State-Trait Anxiety Inventory score >40. Hysteroscopy Visual Analog Scale score was significantly correlated to surgeon experience and to vaginal speculum insertion but not to State-Trait Anxiety Inventory score, ethnicity or abnormal hysteroscopic findings.Conclusion Diagnostic hysteroscopy was mostly perceived as a mild discomfort procedure by most women. Nevertheless, in a considerable number of cases, women perceived hysteroscopy as painful. Pain perception was linked to individual pain threshold and surgeon experience, but not to pre-procedural anxiety state levels, ethnicity or abnormal hysteroscopic findings.