We have extended our previous survey of the association of mitochondrial prevalence in particular tissues with ascites susceptibility in broilers. We previously reported that in breast muscle of 22 week old susceptible line male birds had significantly higher mtDNA copy number relative to nuclear copy number (mtDNA/nucDNA), compared to resistant line male birds. The higher copy number correlated with higher expression of PPARGC1A mRNA gene. Ascites is a significant metabolic disease associated with fast-growing meat-type chickens (broilers) and is a terminal result of pulmonary hypertension syndrome. We now report the mtDNA/nucDNA ratio in lung, liver, heart, thigh, and breast of both genders at 3, and 20 weeks old. At 3 weeks the mtDNA/nucDNA ratio is significantly higher in lung, breast, and thigh for susceptible line males compared to the resistant line males. Conversely, we see the opposite for lung and breast in females. At 20 weeks of age the differences between males from the two lines is lost for lung, and thigh. Although there is a significant reduction in the mtDNA/nucDNA ratio of breast from 3 weeks to 20 weeks in the susceptible line males, the susceptible males remain higher than resistant line males for this specific tissue. We assessed relative expression of five genes known to regulate mitochondrial biogenesis for lung, thigh and breast muscle from males and females of both lines with no consistent pattern to explain the marked gender and line differences for these tissues. Our results indicate clear sex differences in mitochondrial biogenesis establishing a strong association between the mtDNA quantity in a tissue-specific manner and correlated with ascites-phenotype. We propose that mtDNA/nucDNA levels could serve as a potential predictive marker in breeding programs to reduce ascites.