The present disclosure provides a mask assembly, a color filter substrate and a method of manufacturing the same. The mask assembly includes: a first mask formed with a plurality of first black matrix marking mask patterns at a position corresponding to each marking region, a spacing between centers of two first black matrix marking mask patterns that are the closest to each other among the first black matrix marking mask patterns being not less than two times of a spacing between centers of two adjacent sub-pixels located in a same row; and a second mask formed with a plurality of photoresist block mask patterns at a position corresponding to the color filtering region, and further formed with at least two photoresist marking mask patterns, which are located at a position corresponding to each marking region and arranged in a column direction.