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It is generally recognised that electron beam melted (EBM) Ti-6Al-4V alloys exhibit a microstructural gradient along the build direction, but there have been some inconsistent experimental observations and debate as to the origin and magnitude of this effect. Here we present an unambiguous evaluation of this microstructural gradient and associated mechanical property along the EBM build direction on purpose-built round bar RB samples with build height of 380 mm and rectangular plate RP samples with build height of 120 mm. Columnar prior β grain width was found to increase (from 86 ± 38 to 154 ± 56 µm in RB and from 79 ± 34 to 122 ± 56 µm in RP samples) with the build height and the similar increase was also observed for α lath width (from 0.58 ± 24 to 0.87 ± 33 µm in RB and from 1.50 ± 45 to 1.80 ± 49 µm in RP samples). These observations can be attributed to the thermal gradient in the powder bed that produced a cooling rate gradient along the build height. The measured α lath width variation along the build height followed a log-normal distribution. The graded microstructure resulted in a decrease in micro-hardness which correlated very well with the mean α lath width by following a Hall-Petch relation.