Since 1990 there is an upward trend in the incidence of both acquired and congenital syphilis in the Czech Republic. A similar situation exists in other European countries as well. Higher incidence of syphilis is clearly associated with urban agglomerates and sexual tourism destinations. The only way to reduce the number of cases is a consistent application of mandatory preventive and diagnostic measures. These important measures against the spreading of the infection include mandatory serological testing of pregnant women and newborns (from umbilical blood), antibiotic treatment and systematic follow-up of HIV-positive mothers and children. This paper describes the current epidemiological situation of syphilis in the Czech Republic and presents a review of available diagnostic tests and their significance for diagnosis.