Control of visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani primarily relies on chemotherapy using an increasingly compromised repertoire of antileishmanial compounds. For evaluation of novel drugs, the Syrian golden hamster is considered as a clinically relevant laboratory model. In this study, two molecular parasite detection assays were developed targeting cathepsin-like cysteine protease B (CPB) DNA and 18S rRNA to achieve absolute amastigote quantification in the major target organs liver and spleen. Both quantitative PCR (qPCR) techniques showed excellent agreement with a strong correlation with the conventional microscopic reading of Giemsa-stained tissue smears. Using multiple single tissue pieces and all three detection methods, we confirmed homogeneity of infection in liver and spleen and the robustness of extrapolating whole organ burdens from a small single tissue piece. Comparison of pre- and post-treatment burdens in infected hamsters using the three detection methods consistently revealed a stronger parasite reduction in the spleen compared to the liver, indicating an organ-dependent clearance efficacy for miltefosine. In conclusion, this study in the hamster demonstrated high homogeneity of infection in liver and spleen and advocates the use of molecular detection methods for assessment of low (post-treatment) tissue burdens.