Surface asperities play a leading role in determining the fatigue life of as-built Ti-6Al-4 V components manufactured by electron beam powder bed fusion (PBF-EB). Several roughness parameters are available to characterize the surface asperities This study focuses on identifying the surface roughness parameter that correlates best with fatigue life. To this end, several fatigue test specimens were manufactured using the PBF-EB process and utilizing different contour melting strategies, thus producing as-built surfaces with varying roughness. The focus variation microscopy technique was employed to obtain surface roughness parameters for the as-built surfaces. Selected specimens were characterized using x-ray computed tomography (XCT). Tomography can detect surface-connected features obscured by other parts of the surface that are not visible through optical microscopy. The fatigue life of all specimens was determined using four-point bend testing. Through regression model analysis, maximum pit height (Sv) was identified as the statistically significant roughness parameter with the best fit affecting fatigue life. The fracture zone was closely inspected based on the data collected through XCT prior to fatigue tests. This led to another estimate of the worst-case value for the statistically significant roughness parameter Sv. The Sv parameter values obtained from optical microscopy and XCT were used as the initial crack size in a crack growth model to predict fatigue life. It is observed that life estimates based solely on optical measurements of Sv can be overly optimistic, a situation that must be avoided in predictive design calculations.