Zanthoxylum limonella (Dennst.) Alston. (Rutaceae) or Ma-khwaen is one of the medicinal plants in Thai traditional medicine. To investigate the pharmacognostic specifications and chemical constituents of Z. limonella fruits and seeds. Fruits and seeds of Z. limonella were collected from 15 sources throughout Thailand; then examined the pharmacognostic specification following WHO guideline of quality control method for medicinal plant materials. Microscopic determination of Z. limonella powders demonstrated fragment of mesocarp, fragment of brown vitta, oil glands, fragment of endocarp, and endosperm containing oil globule, trichome and pale brown stone cells. Stomatal index and pellucid dots in mm2 were 19.87 and 4.2 respectively. Physico-chemical parameters unveiled that loss on drying, water content, total ash, and acid-insoluble ash should be not >17.90%, 9.18%, 4.50%, and 0.60% of dried weight respectively; while ethanol, water, and hexane extractive values and volatile oil content should be not <2.24%, 2.27%, 1.57% and 9.63% of dried weight respectively. Rf values of thin-layer chromatographic fingerprint of Z. limonella fruits and seeds ethanolic extract were 0.38, 0.45, 0.90, and 0.97 detected ultraviolet (UV) light 254 nm, 0.30, 0.44, 0.67, and 0.77 detected UV light 366 nm, and 0.24, 0.73, 0.78, and 0.93 detected 10% sulfuric acid. There are three main chemical compounds in Z. limonella oil including limonene (43.63%), (+)-sabinene (16.72%), and terpinen-4-ol (10.95%). The result gained from pharmacognostic specifications and chemical fingerprints could be used as standardization data of Z. limonella fruits and seeds to apply or provide for guarantee of quality.