Leishmaniasis is a typical example of a worldwide diffused zoonosis. Geographic distribution depends on the presence of sand fly vectors and animal reservoirs. In Southern Europe, canines are considered the main reservoir of infection, and the phlebotomines are the vectors. In Sicily, as in all Mediterranean areas, sand flies are present almost all year around because the climate permits an uninterrupted lifecycle for the vectors. Visceral leishmaniasis is becoming a real public health concern especially in endemic areas; in fact, it is an opportunistic infection in immunocompromised patients and in HIV-positive subjects. In Italy, the visceral form of the disease is due exclusively to Leishmania infantum ZMON1, and its prevalence is growing. We have developed a highly accurate, reproducible, and sensible real-time polymerase chain reaction (PCR) assay. In a procedure that used a specific couple of primers, a 117-bp fragment was amplified from minicircle kinetoplast DNA (kDNA). The assay was able to detect even a single parasite (200 fg of DNA). In fact, a single parasite contains hundreds of kinetoplast minicircles for each class. We applied a rapid extraction method coupled with the real-time PCR assay. It was not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA, but also more rapid. The assay is useful for the diagnosis of leishmaniasis in dogs and humans, and it facilitates the monitoring of parasite levels during pharmacological treatment.