Gaussian graphical models are semi-algebraic subsets of the cone of positive definite covariance matrices. Submatrices with low rank correspond to generalizations of conditional independence constraints on collections of random variables. We give a precise graph-theoretic characterization of when submatrices of the covariance matrix have small rank for a general class of mixed graphs that includes directed acyclic and undirected graphs as special cases. Our new trek separation criterion generalizes the familiar $d$-separation criterion. Proofs are based on the trek rule, the resulting matrix factorizations and classical theorems of algebraic combinatorics on the expansions of determinants of path polynomials.