Segregation is one of the quality standards that must be monitored during the fabrication and placement of Portland cement concrete. Segregation refers to separation of coarse aggregate from the cement paste, resulting in inhomogeneous mixture. This study introduces a digital imaging based technique to quantify the segregation of Portland cement concrete from 2D digital images of cut sections. In the previous studies, segregation was evaluated based on the existence of coarse aggregate fraction at different geometrical regions of a sample cross section without considering its distribution characteristics. However, it is shown that almost all particle fractions can form clusters and increase the degree of segregation, thus deteriorating the structural performance of concrete. In the proposed methodology, a segregation index is developed by based on the spatial distribution of different size fractions of coarse aggregate within a sample cross section. It is shown that degradation in mixture's homogeneity is controlled by the combined effect of particle distribution and their relative proportions in the mixture. Hence, a segregation index characterizing the mixture inhomogeneity is developed by considering not only spatial distribution of aggregate particles, but also their size fractions in the mixture. The proposed methodology can be successfully used as a quality control tool for monitoring the segregation level in hardened concrete samples.