It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes, but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback and then barcoded for tracking via sequencing (i.e., random deletion library sequencing [RanDeL-seq]). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1's cis and trans elements. The resulting landscape recapitulated HIV-1's known cis-acting elements (i.e., long terminal repeat [LTR], Ψ, and Rev response element [RRE]) and, surprisingly, indicated that HIV-1's central DNA flap (i.e., central polypurine tract [cPPT] to central termination sequence [CTS]) is as critical as the LTR, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼300-bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV's cis-acting regions are larger than the untranscribed (UTR) termini, encompassing a large fraction of the nonstructural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants, enabling discovery of replication mechanisms and development of novel antiviral therapeutics, particularly for emerging viral infections.IMPORTANCE Recent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)-a subset of viral deletion mutants that conditionally replicate. Identifying and engineering DIPs require that viral cis- and trans-acting elements be accurately mapped. Here, we introduce a high-throughput method (random deletion library sequencing [RanDeL-seq]) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central polypurine tract (cPPT), and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.