The incidence of mumps has decreased in many countries since the introduction of vaccination programmes, however, in the past decade a rapid increase in the disease occurrence has been reported worldwide. The reason for this situation is still not clear. We present the results of a serological survey carried out in the Eastern Bohemia Region of the Czech Republic during the years 2008-2012. In total, 2,536 samples of 2,034 patients were examined during the study period. The study cohort was divided into two groups, one consisted of individuals born before the introduction of mandatory vaccination and the other one comprised individuals born after mandatory vaccination started. For the serology analyses the ELISA kits RIDASCREEN Mumpsvirus IgM and IgG (R-Biopharm®, Germany) were used. Out of 2,536 samples (including paired sera), 23.9% (n=606) were positive and 12% (n=304) had equivocal results. Most of the positive samples were obtained from patients aged 17-20 years. Significantly more (p<0.05) positive patients were born after the start of the national vaccination programme (patient group 2) (22.8%) compared to those born before its start (patient group 1) (13.7%). Interestingly, the analysis of data showed that 75.3% of patients falling into group 1 had anti-mumps IgG antibodies, which means that they had contracted mumps, whilst 23.5% of patients of group 2 had undetectable IgG antibodies, even though they should have been vaccinated. The data from our study, with a low number of positive samples in the first years of the study and an increase in the last two years, could suggest the occurrence of outbreaks every 4-6 years.