Fermentation profiling is a common tool to identify Lactobacillus paracasei from other related members of Lactobacillus genus. Because of the proximate biochemical characteristics, identification based on the fermentation pattern within L. casei group (L. casei, L. paracasei and L. rahmnosus) has considerable limitation. On the other hand, members of L. casei group are genetically very closely related and sequencing of signature region is the most reliable and rapid method of differentiation. In this study, instead of sequencing entire 16S or 23S ribosomal RNA gene, we have sequenced 16s-23s rRNA intergenic spacer region and compared them across the members of L. casei group. We also compiled thirteen signature sequences within 16s-23s ISR rRNA gene of L. casei group members. Our isolated strain (L. paracasei ssp. paracasei-1) resembled 100% and 99% similarity when compared with 16s-23s rRNA intergenic spacer region of L. paracasei and L. casei respectively. Our study summarizes that sequencing of short 16s-23s rRNA intergenic spacer region carries great significance in identification of closely related probiotic bacteria such as the members of L. casei group. The findings of this research could be very much helpful for food and pharmaceutical industries who are dealing with probiotic bacteria.