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Brown heartwood rot is an increasingly important problem in southwestern deserts, affecting numerous native tree species, and commercial lemon production. Two fungal pathogens, Coniophora eremophila and Antrodia sinuosa, have been identified as causal agents of brown heartwood rot in citrus. Development of effective disease management strategies relies on the ability to readily identify and distinguish between the pathogens; therefore we have developed molecular markers as an alternative to morphometric methods to differentiate these two economically important pathogens. Random amplified polymorphic DNA analysis identified four primers that revealed fungal polymorphisms. Polymorphic DNA fragments were cloned, sequenced, and used to create a 700 bp sequence characterized amplified region (SCAR) in C. eremophila isolates using sequence-specific primers, Conio7 and Conio8. Three fragments (300, 650, and 1000 bp) were amplified with Conio7 and Conio8 in A. sinuosa isolates. A rapid, small scale method for DNA extraction from fungus was developed to expedite the technique. With this method, the usefulness of the Conio7 and Conio8 was verified using new Coniophora eremophila and An trodia sinuosa isolates from citrus.