There is accumulating evidence that MO increases offspring risk of obesity and cardiovascular disease in later life. Utilizing a well-established (J. Anim. Sci. 2010. 88:3546) ovine model of diet-induced MO from conception through gestation, we reported that gestational d 135 fetal heart left ventricle weight and collagen content were markedly increased by MO. Further, in a Langendorff system, the d 135 fetal heart from MO fetuses could not sustain high work levels (FASEB J. 2010. 24:2066). Here we examined the effect of MO on increasing collagen content and crosslinking in the adult F₁ and F₂ offspring myocardium. Ewes were assigned randomly as controls (CON) and fed at 100% of NRC recommendations or to an MO group fed 150% of NRC from 60 d before conception to term. Left ventricular free wall myocardial samples were collected from adult (22 mo old) male F1 and F2 offspring of CON and MO ewes at necropsy following a 12-week ad lib feeding challenge. Myocardial tissue was either snap frozen in liquid nitrogen for collagen analyses or fixed and paraffin embedded for histological evaluation. Collagen concentration was determined by colorimetry as hydroxyproline equivalents. Collagen crosslinking was also determined by measuring the amount of hydroxylysylpyridinoline by immunoassay (Microvue, PYD EIA Kit, Quidel, San Diego, CA). Data were analyzed by mixed procedures of SAS. Myocardial collagen concentration was greater (P < 0.05) in MOF1 compared with CON F1 offspring (1.73 ± 0.10 vs. 1.42 ± 0.07 µg/mg, n = 6). The differences in OBF1 and CONF1 collagen concentration were confirmed by visualizing trichrome stained sections of myocardial tissue. Myocardial collagen crosslinking was 11% greater (P < 0.05) in OBF1 vs. CONF1 offspring. In contrast, myocardial collagen content and crosslinking did not differ between MOF2 and CONF2 offspring. These data provide evidence for programming of increased myocardial fibrosis in adult MOF1 offspring, which potentially results from MO-induced changes in fetal life and might impair cardiac muscle function in postnatal life.