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Using benthic macrofauna to assess the water resource quality has been proposed because they are intimately related to a living environment. The objective of this study was to observe the benthic diversity and the biotic index, BMWPthai score (Biological Monitoring Working Party) and ASTPthai (Average Score Per Taxa) of macrofauna of the Pasak River, Ayutthaya province. Results from the study showed that the discovery of macrofauna was totally found 23 species in 17 families. The dominant macrofauna in relation to the pollution-tolerant species, including aquatic oligochaete (family Tubificida), polychaete (families Nephtyidae and Nephtyidae), gastropod (family Viviparidae), bivalve (family Corbiculidae), and insect larvae (family Chironomidae) were discovered. According to BMWPthai Score and ASPTthai Score, it was indicated that the Pasak River was considered to be a poor water quality depending on the standard criteria of surface water quality category 4. This research warranted that BMWPthai and ASPTthai Scores can be used to assess the water quality, which is associated with the consideration of benthic species composition, diversity index and sediment organic matter.