Current practice of determining river water quality in Malaysia is based mainly on physicochemical components. Perhaps, owing to the lack of information on habitat requirements and ecological diversity of aquatic macroinvertebrates and on unearthly taxonomic key of benthic macroinvertebrates in this region makes it less popular than conventional methods. The study took place in three rivers in the state of Johor, Southern Peninsula of Malaysia, which exhibited different degrees of disturbances and physical properties, namely Sungai Ayer Hitam Besar, Sg Berasau, and Sg Mengkibol. Benthic macroinvertebrates were sampled using rectangular dipnet with frame dimension 0.5 m × 0.3 m. Although physicochemical elements such as water temperature, pH, and dissolved oxygen (DO) were measured using a YSI Professional Plus handheld multiparameter instrument, other parameters such as biochemical oxygen demand (BOD 5 ), chemical oxygen demand (COD), total suspended solid (TSS), and ammoniacal nitrogen (NH 3 N) were tested using the procedure of APHA Standard Method. The study found that the status of water quality varies among the three rivers. A multivariate analysis, the canonical correspondence analysis (CCA), was applied to elucidate the relationships between biological assemblages of species and their environment using PAST (version 2) software. The present findings reveal that humaninduced activities are the ultimate causes of the alteration in macroinvertebrate biodiversity.