A novel, genetically-stabilized type 2 oral polio vaccine (nOPV2), developed to assist in the global polio eradication program, was recently the first-ever vaccine granted Emergency Use Listing by the WHO. Lot release tests for this vaccine included-for the first time to our knowledge-the assessment of genetic heterogeneity using next-generation sequencing (NGS). NGS ensures that the genetically-modified regions of the vaccine virus genome remain as designed and that levels of polymorphisms which may impact safety or efficacy are controlled during routine production. The variants present in nOPV2 lots were first assessed for temperature sensitivity and neurovirulence using molecular clones to inform which polymorphisms warranted formal evaluation during lot release. The novel use of NGS as a lot release test required formal validation of the method. Analysis of an nOPV2 lot spiked with the parental Sabin-2 strain enabled performance characteristics of the method to be assessed simultaneously at over 40 positions in the genome. These characteristics included repeatability and intermediate precision of polymorphism measurement, linearity of both spike-induced and nOPV2 lot-specific polymorphisms, and the limit-of-detection of spike-induced polymorphisms. The performance characteristics of the method met pre-defined criteria for 34 spike-induced polymorphic sites and 8 polymorphisms associated with the nOPV2 preparation; these sites collectively spanned most of the viral genome. Finally, the co-location of variants of interest on genomes was evaluated, with implications for the interpretation of NGS discussed.