The microhabitat preference of zebrafish, Danio rerio from Karala river was investigated. The availability/ preference of microhabitat variables such as (i) distance from the stream bank (ii) water column depth (iii) mesohabitat (iv) water velocity (v) substratum and (vi) subaquatic vegetal cover were quantified based on underwater observations covering a total of 400 m2 of stream area. Microhabitat selectivity was analyzed by comparing the microhabitat availability in the study site and the microhabitat used by fish. Data availability and microhabitats usage pattern were used for calculating habitat availability/preference of D. rerio. In order to test the microhabitat preference of D. rerio the Principal Component Analysis (PCA) and Canonical Correspondence Analysis (CCA) were applied. In PCA the first three components with higher Eigenvalue accounted for about 98.46% of the total variance. The first component axis alone explained 86.33% of the variance with an Eigenvalue of 5.3 with high loadings (>0.7). CCA revealed a clear separation of the D. rerio along the microhabitat variables. Among the thirty two microhabitat variables, the forward selection procedure was found to be positively significant with eighteen microhabitat variables P<0.05. The preference of D. rerio in the studied streams viz: pool, plunge pool and isolated pool; slow flow, moderate flow and fast flow; and the substratum parameters: sand, gravel and leaf litter; depth parameters like Depth 1, Depth 2, Depth 3 and Depth 5; fish cover parameters such as root undercut and distance from bank parameters such as Distance from bank 1, Distance from bank 2 and Distance from bank 3. CCA revealed a clear distribution pattern of the D. rerio along the microhabitat variables.