Profile-based analysis of multiple sequence alignments (MSA) allows for accurate comparison of protein families. Here, we address the problems of detecting statistically confident dissimilarities between (1) MSA position and a set of predicted residue frequencies, and (2) between two MSA positions. These problems are important for (i) evaluation and optimization of methods predicting residue occurrence at protein positions, (ii) detection of potentially misaligned regions in automatically produced alignments and their further refinement, and (iii) detection of sites that determine functional or structural specificity in two related families.