As a first step in assessing the viability of periphyton-based shrimp production in lined pond aquaculture systems, the effects of natural substrates (split bamboo poles and coconut coir) on development of periphyton were evaluated. The periphyton biomass was recorded at 0-40 cm depth, 40 - 80 cm depth and 80 - 120 cm for all the substrates. The periphyton biomass, in terms of dry matter (DM) from coconut coir was comparatively higher (3.4995 ± 0.31201 mg/cm2) than the split bamboo poles (3.2011 ± 0.30437 mg/cm2) during the experiment. One way ANOVA of the different data affirmed that significant difference (P < 0.05) was observed in the dry matter content of the periphyton among the substrates. As per the Duncan's Multiple Range Test, dry matter content of periphyton recorded for coconut coir and split bamboo poles showed that there was no significant difference (P < 0.05) between them. Student's 't' test analysis of the data relating to periphyton biomass for split bamboo pole (P<0.05) and coconut coir (P<0.003) recorded at different depths indicated that upper 0-40 cm depth had higher values than the other depths. Further, among the two substrate were used, coconut coir (3.4995 ± 0.31201 mg/cm2) was found to be best substrate than split bamboo pole for periphyton attachment based on the periphyton biomass.