Among additive manufacturing technologies, Laser Powder Bed Fusion (L-PBF) is considered the most widespread layer-by-layer process. Although the L-PBF, which is also called as SLM method, has many advantages, several challenging problems must be overcome, including part positioning issues. In this study, the effect of part positioning on the microstructure of the part in the L-PBF method was investigated. Five Ti6Al4V samples were printed in different positions on the building platform and investigated with the aid of temperature, porosity, microstructure and hardness evaluations. In this study, martensitic needles were detected within the microstructure of Ti6Al4V samples. Furthermore, some twins were noticed on primary martensitic lines and the agglomeration of β precipitates was observed in vanadium rich areas. The positioning conditions of samples were revealed to have a strong effect on temperature gradients and on the average size of martensitic lines. Besides, different hardness values were attained depending on sample positioning conditions. As a major result, cooling rates were found related to positions of samples and the location of point on the samples. Higher cooling rates and repetitive cooling cycles resulted in microstructures becoming finer and harder.