Sequence data is becoming increasingly accessible creating a critical need for end-user tools that leverage the information to facilitate biological research. The computational analysis tool for high-throughput identification of restriction enzyme sites, CisSERS, was developed to facilitate post-sequence acquisition applications such as selective transcriptome sequencing and molecular marker development. The processing power of CisSERS is limited only by the computer resources utilized to evaluate the sequences. Single or multiple sequences in FASTA format can be processed with all available, custom selected, or user input restriction enzymes for a myriad of biological research projects. Users can select entire genomic or transcriptomic sequences, or regions of sequences between, or from, the 5’ and 3’ ends to be processed. CisSERS analysis enables enrichment of transcripts for 5’ or 3’ ends for subsequent UTR sequencing by identifying common restriction sites for the majority of sequences. Research project strategies are facilitated via a multi-comparative digital gel application allowing the user to visualize predicted restriction sites on a single sequence with multiple enzymes or multiple sequences with a single enzyme. Determination of restriction sites for cloning, sequence verification and screening, vector construction, DNA fingerprinting and primer design can also be accomplished. CisSERS generated fingerprints for applications such as RFLP has been tested utilizing a HindIII digest of lambda phage. Further, a CisSERS-derived CAPS marker for a gene of interest in apple was successfully verified by gel electrophoresis. The availability of CisSERS will bridge the gap between data generation and data utilization in biological research.