Background Suspected penicillin allergy (Pen-A) is often not verified or excluded by diagnostic testing. Objective To assess the prevalence and impact of Pen-A registration in a Dutch University Medical Center. Methods In a prospective matched cohort study, all admitted patients (July 2013-July 2014) who underwent a pharmacotherapeutic interview were selected. Patients with a registered Pen-A were matched on age, sex, and department of admission with up to 3 patients without a registered Pen-A. Relative risks (RRs) of receiving a reserve antibiotic, death during hospitalization, and rehospitalization were compared in the 2 cohorts. The number and type of antibiotics prescribed during admission and duration of hospitalization were compared. Results Of 17,959 patients, 1010 (5.6%) patients (66.7% women; median age, 55 years) had a Pen-A registration. These patients had a higher risk of receiving reserve antibiotics (RR, 1.38; 95% CI, 1.22-1.56) and of being rehospitalized within 12 weeks (RR, 1.28; 95% CI, 1.10-1.49). A significantly larger proportion of Pen-A registered patients received reserve antibiotics such as tetracyclines (1.8% vs 0.8%), macrolides/lincosamides/streptogramins (12.5% vs 4.9%), and quinolones (7.9% vs 4.3%) or received 2 or more types of antibiotics during hospitalization (21.7% vs 16.9%). Conclusions Prevalence of Pen-A registration in hospitalized patients is high, has high impact on antibiotic prescribing, and is associated with a higher risk of readmission. Verification of the Pen-A in hospitalized patients might restrict the use of reserve antibiotics and improve patient outcome.