Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, we analyzed if PDAC resistance to VSV could be decreased by directed viral evolution using our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC. Each virus encodes VSV matrix protein with ∆M51 mutation (M-∆M51) and one of two versions of a functional human tumor suppressor p53 fused to a fluorescent reporter protein eqFP650. Each virus was serially passaged 32 times (accounts for more than 60 viral replication cycles) on either SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and another PDAC cell line AsPC-1 (moderately resistant to VSV), while remaining highly attenuated in non-malignant cell lines. Surprisingly, two identical VSV glycoprotein (G) mutations, E238K and K174E were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-∆M51 protein and no deletions or mutations were found in the p53 or eqFP650 portions of virus-encoded transgenes in any of the passaged viruses. These findings demonstrate long-term genomic stability of complex VSV recombinants encoding large transgenes, and support further clinical development of oncolytic VSV recombinants as safe cancer therapeutics.