Molecular characterization of crops improved through biotechnology has traditionally been conducted using Southern blot analysis which has been used to determine T-DNA copy number, the presence or absence of backbone (sequence outside of the T-DNA) and to demonstrate generational stability of the T-DNA insert. The advancement of high-throughput DNA sequencing (HTS) technology allows efficient characterization of the transgene incorportated into the genome of the plant by rapidly sequencing the entire plant genome. By combining NGS (Next Generation Sequencing) technologies with bioinformatic methods that identify the T-DNA insert derived from the plasmid vector and genome-T-DNA junction sequences, it has been shown that conclusions equivalent to those of a Southern blot are readily obtained. NGS is done at sufficient coverage depth (>75x) across the entire genome. By mapping the sequence reads to the plasmid vector, and identifying the number of unique junctions, we can confirm insert number, copy number, absence of backbone, across multiple generations. With the widespread availability of NGS and steadily decreasing costs it is likely that academia and industry will fully transition to NGS-based molecular characterizations in the near future.