Dearth of genomic resources particularly, microsatellite markers in nutritionally and commercially important fruit crop, guava necessitate the development of the novel genomic SSR markers through the library enrichment techniques. Three types of 3' -biotinylated oligonucleotide probes [(CT)14, (GT)12, and (AAC)8] were used to develop microsatellite enriched libraries. A total of 153 transformed colonies were screened of which 111 positive colonies were subjected for Sanger sequencing. The clones having more than five motif repeats were selected for primer designing and a total of 38 novel genomic simple sequence repeats could be identified. The g-SSRs had the motif groups ranging from monomer to pentamer out of which dimer group occurred the most (89.47%). Out of 38 g-SSRs markers developed, 26 were found polymorphic, which showed substantial genetic diversity among the guava genotypes including wild species. The average number of alleles per locus, major allele frequency, gene diversity, expected heterozygosity and polymorphic information content of 26 SSRs were 3.46, 0.56, 0.53, 0.29 and 0.46, respectively. The rate of cross-species transferability of the developed g-SSR loci varied from 38.46 to 80.77% among the studied wild Psidium species. Generation of N-J tree based on 26 SSRs grouped the 40 guava genotypes into six clades with two out-groups, the wild guava species showed genetic distinctness from cultivated genotypes. Furthermore, population structure analysis grouped the guava genotypes into three genetic groups, which were partly supported by PCoA and N-J tree. Further, AMOVA and PCoA deciphered high genetic diversity among the present set of guava genotypes including wild species. Thus, the developed novel g-SSRs were found efficient and informative for diversity and population structure analyses of the guava genotypes. These developed novel g-SSR loci would add to the new genomic resource in guava, which may be utilized in genomic-assisted guava breeding.