The Indian mackerel, Rastrelliger kanagurta, is a commercially valuable fish across the Indonesia archipelagic waters. The species was managed and assessed under a Fisheries Management Areas (FMAs) management concept, that establish based on the similarity of environmental characteristics. Despite its important role in the fisheries industry, information regarding stock structures for management and conservation purposes within a single FMA is still not known. This study aimed to identify the Indian mackerel stock by otolith shape variability along the southern coast of Java-Bali. Otolith samples were collected in 2016 and 2018 from four fishing ports: Palabuhanratu, Pacitan, Muncar, and Kedonganan. Otolith outline was reconstructed by using ten levels and 64 Wavelet shape coefficients. The average of otolith shape for each location was visualized in x- and y-axis matrix overlaid to 0-360° angle correspond to morphological features of the otolith. Statistical analysis using a series of ANOVA-like permutation test followed by a cluster analysis using Canonical Analysis of Principal Coordinates (CAP), and Linear Discriminant Analysis (LDA) was applied to differentiate otolith variation between locations. The results showed significant differences in otolith shape, especially in four otolith morphological parts, namely the excisura major, antirostum, pararostrum, and postrostrum. Also, the variation of otolith shape was significantly different between localities (p=0.001). The LDA using cross-validation procedures correctly classified individual samples back to their original group with a success rate was ranged from 44.26 to 82.61%. These results indicate the existence of two major groups of R. kanagurta contributed to the stock that can have implications for providing more precisely of stock assessment and its management scenario for sustainable fisheries.