Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant morbidity and mortality in tropical and sub-tropical areas worldwide. Despite extensive studies, there are significant gaps in our understanding of the exact features of the viral genome and its encoded proteins that are critical to the viral replication cycle. Here we subjected an infectious DENV genome to unbiased insertional mutagenesis and employed next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in Huh-7.5 hepatic cell culture and following mutant viral library passage onto Vero and C6/36 cell lines. This revealed that broad regions within capsid, NS1 and the 3’UTR were most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Exploiting this information we generated infectious reporter viruses, including a variants that were tailored for live cell imaging of NS1 localization and traffic, electron microscopy- and super resolution microscopy-based studies of NS1 localization and sensitive luciferase-based studies of NS1 accumulation, secretion and interactions. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and application and in future antiviral strategies.