Mapping the longitudinal relaxation time (T(1)) of brain tissue is of great interest for both clinical research and MRI sequence development. For an unambiguous interpretation of in vivo variations in T(1) images, it is important to understand the degree of variability that is associated with the quantitative T(1) parameter. This paper presents a general framework for estimating the uncertainty in quantitative T(1) mapping by combining a slice-shifted multi-slice inversion recovery EPI technique with the statistical wild-bootstrap approach. Both simulations and experimental analyses were performed to validate this novel approach and to evaluate the estimated T(1) uncertainty in several brain regions across four healthy volunteers. By estimating the T(1) uncertainty, it is shown that the variation in T(1) within anatomic regions for similar tissue types is larger than the uncertainty in the measurement. This indicates that heterogeneity of the inspected tissue and/or partial volume effects can be the main determinants for the observed variability in the estimated T(1) values. The proposed approach to estimate T(1) and its uncertainty without the need for repeated measurements may also prove to be useful for calculating effect sizes that are deemed significant when comparing group differences.