A wide range of nutrients and health-promoting non-nutrient components in mushrooms are a subject of international research, but specific reference materials to facilitate comparison of results among laboratories are lacking. Commercially available food matrix reference materials do not contain components unique to mushrooms (e.g., ergosterol, vitamin D 2 , chitin, beta-glucans, agaritine, ergothioneine). A Mixed Mushroom Control Material (CM) (homogeneous mixture of 15 types of mushrooms) was prepared and characterized for selected components, including proximates (moisture, protein, ash), total folate, folate vitamers, ergosterol, ergosterol metabolites, vitamin D 2 (ergocalciferol), amino acids, total dietary fiber, agaritine, elements (sodium, potassium, phosphorous, magnesium, calcium, iron, copper, manganese, zinc), riboflavin, niacin, thiamin, vitamin B6, pantothenic acid). Subsamples of the CM are available to qualified laboratories from the Food Analysis Laboratory Control Center at Virginia Tech (Blacksburg, VA, USA), to be assayed concurrently with mushroom samples for which food composition data will be published along with results for the CM. Implementation of this CM should facilitate comparison of published data on mushroom composition and health benefit among species, and biodiversity within species by serving as common control sample that allows the separation of analytical variability from true differences in sample composition determined at different laboratories.