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In 2012, an ongoing outbreak of diphtheria in Indonesia was focused in the province of East Java. There was a need to assess vaccine coverage and immunity gaps in children. We conducted a cross-sectional seroprevalence and vaccine coverage survey of children 1-15 years of age in two districts of East Java: one of high incidence (on the island of Madura) and one of low incidence (on the mainland). From each district we sampled 150 children (10 children per year of age). Sera and throat swabs were taken to determine immunity and carriage status. Immunity was defined as ≥0.1 international unit/ml of antibody to diphtheria toxin. A total of 297 children were selected to participate in the study. DTP3 coverage was significantly lower (P<0.001) in the high incidence district compared with the low (57%, 95% confidence interval [CI]: 36-78 vs. 97%, 95% CI: 93-100). Despite this higher vaccine coverage, seroprevalence of immunity was lower in the low incidence district compared with the high (71%, 95% CI: 63-80 vs. 83%, 95% CI: 76-90). Immunity in the high incidence district was associated with increased age, increased prevalence of toxigenic Corynebacterium diphtheriae carriers and with receipt of multiple (and likely more recent) boosters. Significant variation exists in vaccine coverage and seroprevalence of immunity to diphtheria in East Java. Immunity in high incidence districts is likely due to natural immunity acquired through exposure to toxigenic C. diphtheriae. Booster vaccines are essential for achieving protective levels of immunity.