Excessive fear generalization has been associated with pathological anxiety, including posttraumatic stress disorder (PTSD). However, studies investigating the longitudinal relationship between generalization and the development of anxiety symptomatology are scarce. This study aims to test the predictive value of fear generalization for PTSD symptoms in a high-risk profession sample and to explore the relationship between generalization and neuroticism, which are both linked to PTSD. Longitudinal data from a multi-wave study in 529 Dutch fire-fighters were used. Fear generalization, PTSD symptoms and neuroticism were assessed at baseline. PTSD symptoms were reevaluated at six, 12, 18, and 24 months. Generalization was assessed in a differential conditioning paradigm by measuring expectancies of an aversive outcome when presented with stimuli similar to previously conditioned stimuli. Higher expectancy ratings towards stimuli most similar to safety signals predicted PTSD symptoms at follow-up after controlling for baseline PTSD symptoms, whereas higher expectancy ratings towards stimuli most similar to danger signals was associated with neuroticism. Neuroticism weakened the predictive power of fear generalization when considered simultaneously. These findings suggest that heightened fear generalization is associated with the development of anxiety and trauma-related symptoms. Targeting problematic fear generalization may be a promising intervention approach. •Enhanced fear generalization predicted PTSD symptom development in fire-fighters.•Responses to generalized stimuli similar to the safe cue were linked to PTSD.•Higher neuroticism was associated with enhanced fear generalization.•Neuroticism weakened the predictive power of fear generalization for PTSD symptoms.