Although Denmark has a low hepatitis B virus (HBV) prevalence, HBV transmission has been reported in Danish day-care centres. The aim of this study was to validate saliva anti-HBc testing as a method for HBV screening, the applicability of saliva sampling to pre-school children, and to determine the HBV prevalence in Danish day-care centres with a high proportion of immigrants. For validation, paired saliva and plasma samples were obtained from blood donors and injecting drug users. Employees and children in day-care centres with a high proportion of immigrant children were offered saliva screening followed by blood test if positive. The specificity and sensitivity of anti-HBc tests on saliva was 100% (102 blood donors and four injecting drug users) and 85.9% (61 of 71 anti-HBc-positive injecting drug users), respectively. In all samples from HBsAg (n = 7) or anti-HBc IgM-positives (n = 9), anti-HBc was detected in saliva. Adequate saliva samples were obtained from 93% (588/634) of children and 100% (166/166) of employees participating in the day-care centre survey. Among children 55% were of non-Scandinavian origin and only one (0.2%, 95% CI [0.0; 1.0]) was HBV positive. Among employees the corresponding values were 22% and 7 (4.2%). The positive predictive value of the saliva test was 25% (1/4) among children and 88% (7/8) among adults. In conclusion, saliva testing is feasible for HBV screening among children in low prevalence populations, but any anti-HBc reactivity should be confirmed by plasma analysis. The HBV prevalence in pre-school children in Denmark is low even among immigrants from endemic areas.