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Black pepper (Piper nigrum) is a vital spice crop with uses ranging from culinary to pharmacological applications. However, limited genetic information has constrained the understanding of the molecular regulation of flower and fruit development in black pepper. In this study, a comparison among three different black pepper varieties, Semengok Aman (SA), Kuching (KC), and Semengok 1 (S1), with varying fruit characteristics was used to provide insight on the genetic regulation of flower and fruit development. Next-generation sequencing (NGS) technology was used to determine the flower and fruit transcriptomes by sequencing on an Illumina HiSeq 2500 platform followed by de novo assembly using SOAPdenovo-Trans. The high-quality assembly of 66,906 of unigenes included 64.4% of gene sequences (43,115) with similarity to one or more protein sequences from the GenBank database. Annotation with Blast2Go assigned 37,377 genes to one or more Gene Ontology terms. Of these genes, 5,874 genes were further associated with the biological pathways recorded in the KEGG database. Comparison of flower and fruit transcriptome data from the three different black pepper varieties revealed a large number of DEGs between flower and fruit of the SA variety. Gene Ontology (GO) enrichment analysis further supports functions of DEGs between flower and fruit in the categories of carbohydrate metabolic processes, embryo development, and DNA metabolic processes while the DEGs in fruit relate to biosynthetic process, secondary metabolic process, and catabolic process. The enrichment of DEGs in KEGG pathways was also investigated, and a large number of genes were found to belong to the nucleotide metabolism and carbohydrate metabolism categories. Gene expression profiling of flower formation-related genes reveals that other than regulating the flowering in black pepper, the flowering genes might also be implicated in the fruit development process. Transcriptional analysis of sugar transporter and carbohydrate metabolism genes in different fruit varieties suggested that the carbohydrate metabolism in black pepper fruit is developmentally regulated, and some genes might serve as potential genes for future crop quality improvement. Study on the piperine-related gene expression analysis suggested that lysine-derived products might present in all stages of fruit development, but the transportation was only active at the early stage of fruit development. These results indicate several candidate genes related to the development of flower and fruit in black pepper and provide a resource for future functional analysis and potentially for future crop improvement. [ABSTRACT FROM AUTHOR]