We performed a four year study to investigate the Visceral Leishmaniasis (VL) cases in, Turkey. Fifty-nine patients with suspected VL from Istanbul were included in this work. Bone marrow and blood samples of these patients were tested for possible VL infection using several methods including serological tests, microscopy, PCR. Nineteen (32.2%) patients had positive results for VL after one or more of the tests performed, while only 7 patients (11.8%) had positive results with all the tests including Giemsa stain. Four (6.8%) patients had negative results based on all the serological tests performed except for positive results with Giemsa stain, culture and PCR. The other 4 (6.8%) patients had positive results with Formol-gel, ELISA IgG (> 1.1 ISR) and IFAT IgG, (> 1/256) but negative results were obtained with direct microscopic examination, culture and PCR. Using PCR Leishmania infantum DNA was detected in 11(18.6%) of the (Leishmania) cultures originated from the bone marrow samples. Plasmodium vivax was found in 2 (3.4%) patients and leptospira was detected in 1 (1.7%) patient. One (1.7%) patient was diagnosed with Pneumonia (Streptococcus pneumoniae). Forty (67.8%) patients had negative results after direct microscopic examination, culture, serological tests and PCR. The kappa coefficients k = 0.80 k = 1.00, k = 0.51, k = 0.55 and k = 0.45 were evaluated for PCR and direct microscopic examination, PCR and culture, PCR and ELISA, PCR and IFAT and PCR and Formol-Gel, as perfect agreement, perfect agreement, moderate agreement and moderate agreement fair moderate, respectively. The probability values (p) for comparisons of all the above tests with PCR showed a significant correlation (p < 0.000) In conclusion, we found that no single method alone was sufficient enough to diagnose VL accurately; however, combined with PCR, all these methods can reveal better and sensitive results ultimately leading to a correct diagnosis. We also suggest that PCR has to be applied with other laboratory diagnostic tests in order to increase the sensitivity in diagnosis and decrease the possible defects in diagnosis.