Guava (Psidium guajava L.), a rich source of nutrients, is an important tropical and subtropical fruit of the Myrtaceae family and exhibits magnificent diversity. Genetic diversity analysis is the first step toward the identification of parents for hybridization, genetic mapping, and molecular breeding in any crop species. A diversity analysis based on whole-genome functional markers increases the chances of identifying genetic associations with agronomically important traits. Therefore, here, we sequenced the genome of guava cv. Allahabad Safeda on an Illumina platform and generated a draft assembly of ~304 MB. The assembly of the Allahabad Safeda genome constituted >37.95% repeat sequences, gene prediction with RNA-seq data as evidence identified 14,115 genes, and BLAST n/r, Interproscan, PfamScan, BLAST2GO, and KEGG annotated 13,957 genes. A comparative protein transcript analysis of tree species revealed the close relatedness of guava with Eucalyptus. Comparative transcriptomics-based SSR/InDel/SNP-PCR ready genome-wide markers in greenish-yellow skinned and white fleshed-Allahabad Safeda to four contrasting cultivars viz apple-color-skinned and white-fleshed-Lalima, greenish-yellow-skinned and pink-fleshed-Punjab Pink, purple-black-skinned and purple-fleshed-Purple Local and widely used rootstock-Lucknow-49 were developed. The molecular markers developed here revealed a high level of individual heterozygosity within genotypes in 22 phenotypically diverse guava cultivars. Principal coordinate, STRUCTURE clustering, and neighbor-joining-based genetic diversity analysis identified distinct clusters associated with fruit skin and flesh color. The genome sequencing of guava, functional annotation, comparative transcriptomics-based genome-wide markers, and genetic diversity analysis will expand the knowledge of genomes of climacteric fruits, facilitating trait-based molecular breeding and diversifying the nutritional basket.