Although different types of powder feedstock are used for additive manufacturing via laser powder bed fusion (L-PBF), limited work has attempted to directly compare the microstructure and mechanical behavior of components manufactured from those powder feedstock. This work investigated the microstructure, phase composition, melt pool morphology, and mechanical properties of a prealloyed Ti-35Nb alloy manufactured using L-PBF and compared these to their counterparts produced from elemental powder mixture. The samples manufactured from the powder mixture are composed of randomly distributed undissolved Nb in the α/β matrix, resulting from the unstable melt pool during the melting of the powder mixture. By contrast, parts produced from prealloyed powder display a homogeneous microstructure with β and α″ phases, owing to the full melting of prealloyed powder, therefore, a more stable melt pool to achieve a homogeneous microstructure. The Ti-35Nb manufactured from prealloyed powder exhibits large tensile ductility (about 10 times that of the counterparts using mixed powder), attributed to the high homogeneity in microstructure and chemical composition, strong interface bonding, relatively low oxygen content, and the existence of a large amount of β phase. This work sheds insights into understanding the effect of powder feedstock on the melt pool stability therefore the microstructure and mechanical behavior of the resultant parts.