Abstract Body: The adult mammalian heart has limited regenerative capacity due in part to the low level of diploid cardiomyocytes (CMs). Multiple polymorphic genes determine the final level of diploid CMs, however, the relevant genes need to be identified. There is a large variation in CM ploidy between the highly related sister strains BALB/cJ and BALB/cByJ. We undertook an RNA-Seq comparison of neonatal ventricular heart tissue from BALB/cJ and BALB/cByJ to identify gene expression differences that might underlie this difference in CM ploidy. Cytohesin-1 (Cyth1) and Dynein heavy chain 17 (Dnah17) were particularly different in expression: in BALB/cJ (as in most other mouse strains), Cyth1 is expressed in the heart and Dnah17 isn’t expressed, whereas the opposite profile (Cyth1 off, Dnah17 on) was observed in BALB/cByJ heart. These two genes are immediately adjacent to each other in the mouse genome. We found that the basis of differential expression is a large deletion in the locus of BALB/cByJ mice spanning from the Cyth1 first intron to shortly downstream of the last Cyth1 exon. This creates a complete null Cyth1 allele and at the same time results in ectopic transcription of the downstream gene Dnah17, although this transcript is unlikely to encode a protein. Cyth1 encodes a guanine nucleotide exchange factor that was a plausible candidate for being involved in CM ploidy. Using the natural Cyth1 mutant locus plus an engineered null allele, we found no difference in CM ploidy in the absence of Cyth1 function on a cJ-cByJ F1 background or on an inbred C57BL/6J background. This study successfully identified a natural loss-of-function variant in the Cyth1 gene between BALB/cJ and BALB/cByJ but showed that this does not impact CM ploidy, at least by itself. It may underlie other phenotypic differences between these two strains.