The DNA content of some Zea mays chromosomes has been measured by flow karyotyping because similar chromosomes could not been discriminated. This technique requires suspensions with a high amount of intact chromosomes and protocols that provide enough resolution to quantify the DNA content of individual chromosomes. In order to measure the DNA content of A and B chromosomes of Z. mays ‘Black Mexican Sweet Corn’, we associated cytogenetic, flow and image cytometry tools. The cytogenetic methodology provided slides with A and B chromosomes adequate for karyogram assembly. The flow cytometry histograms exhibited distinct G0/G1 and G2 peaks for ‘Black Mexican Sweet Corn’ with (sample) and without (standard) B chromosomes. As a result, the nuclear DNA content of five plants showing B chromosomes was quantified. The nuclear genome size value was proportionally distributed according to the integrated optical density value, which was measured for individual chromosomes by image cytometry. Then, the DNA content of each A and B chromosome was calculated in picograms and base pairs. Based on qualitative (cytogenetic) and quantitative (flow and image cytometry) analyses, it was possible to discriminate and characterize the A and B chromosomes of ‘Black Mexican Sweet Corn’.