This study aimed to standardize qPCR techniques using these molecular markers kDNA and 18S rDNA across three sample types: peripheral blood, guanidine-treated blood, and tissue. The secondary objective is to evaluate the performance of 18S rDNA target in samples from 46 patients with confirmed tegumentary leishmaniasis. After obtaining the standard curve from reference strains with Leishmania, qPCR curves were standardizations and the Cts results of the patient samples were described using abstract measures. Specific specification equations (EEG) with normal distribution and identity link function were constructed to compare each type of clinical sample. To identify the differences among samples and techniques, multiple comparisons with Bonferroni post-test was performed. The kDNA and 18S rDNA demonstrated high sensitivity, detecting as few as 10⁻ parasites/mL. However, 18S rDNA showed limited species discrimination. qPCR performance was evaluated using blood and tissue samples, showing a sensitivity of 54.2% in blood, 12.5% in guanidine-treated blood, and 86.4% in tissue. qPCR agreement with the 18S rDNA target with the three types of samples, positive and negative, in relation to screening were 56.2% in blood, 31.8% in guanidine- blood and tissue 78.6%. As for true positives (PPV), tissue samples presented a probability percentage of individuals being sick of 86.4%, while in blood it was 81.3%. The results underscore the importance of molecular diagnostics in blood samples, improving the accuracy and monitoring of tegumentary leishmaniasis.