Lyme borreliosis (LB) is an endemic disease in adults in Western countries. Although children may also be infected, pediatric studies on LB are scarce. This study aims to estimate the incidence of LB among children with a clinical suspicion for Lyme in a tertiary referral center in the Netherlands. Patient chart data on medical history, clinical signs and symptoms, diagnostic test results and diagnoses were collected using standardized case record forms. Patients were categorized based on clinical and laboratory findings using a modified, previously published classification system. We included 325 children, with a median age of 11.9 years, of whom 61.8% were female. LB was diagnosed in 38 of the referred children (11.7%). However, of the 85 patients who were specifically referred to the Lyme clinic, 28 (32.9%) were diagnosed with LB. Of the specifically referred Lyme-positive patients, 11 (39.3%) had a definitive LB diagnosis. Twelve children had a posttreatment LB syndrome. In line with previous reports in adults, only a small proportion of children referred with a suspicion of LB were diagnosed with definite or probable LB, which illustrates the difficulty in diagnosing LB by the general practitioner or pediatrician in a district hospital.