Background: We investigated breed and gender variations in the compositions of long-chain (≥ C20) omega-3 polyunsaturated fatty acids (LC omega-3 PUFA), fat melting point (FMP) and intramuscular fat (IMF) contents in biopsy samples of the M. longissimus dorsi muscle of grazing beef cattle. The hypothesis that biopsy compositions of health-beneficial LC omega-3 PUFA, FMP and IMF in a pasture-based production system will vary with breed, was tested. Muscle biopsies were taken from 127 yearling pasture-based Angus, Hereford, and Wagyu heifers and young bulls exclusive to the Australian Bowen Genetics Forest Pastoral breeding stud averaging 12 ± 2.43 months of age and under the same management routine. Results: Breed had a significant influence on IMF, FMP, and the compositions of oleic acid, α-linolenic acid (ALA), eicosapentaenoic (EPA), docosahexaenoic (DHA), docosapentaenoic (DPA), and total EPA + DHA + DPA in the M. longissimus dorsi muscle biopsies (P ≤ 0.03). The Wagyu breed had the highest (11.1%) and Hereford the lowest (5.9%) IMF (P = 0.03). The reverse trend was observed in FMP values where the Hereford breed had the highest (55 °C), Angus intermediate (46.5 °C), and Wagyu the lowest (33 °C) FMP. The Wagyu and Angus breeds had similar oleic fatty acid (18:1n-9) content, while the Hereford breed had the lowest (P < 0.01). The highest ALA, DPA, total EPA + DHA, total EPA + DHA + DPA and total ALA + EPA + DHA + DPA contents were detected in the Wagyu breed (P ≤ 0.03). The Hereford had similar EPA and DPA contents to the Angus (P ≥ 0.46). Total EPA + DHA + DPA contents in Wagyu, Angus, and Hereford were 28.8, 21.5, and 22.1 mg/100g tissue (P = 0.01), respectively. Sex was an important source of variation that influenced LC omega-3 PUFA composition, FMP and IMF, where yearling heifers had higher IMF (11.9% vs 5.3%), lower FMP (33°C vs 37°C), and higher LC omega-3 PUFA than bulls. Conclusion: All the results taken together indicate that the Wagyu breed at 28.8 mg/100g tissue, was the closest to meeting the Australia and New Zealand recommended source level threshold of 30 mg/100g tissue of health-beneficial ≥ C20 omega-3 FA content. Since gender was a significant determinant of LC omega-3 PUFA composition, IMF content and FMP, it should be factored into enhancement strategies of healthy meat eating quality traits in grazing cattle. These findings also suggest that the Bowen Genetics Forest Pastoral beef cattle studs are important sources of LC omega-3 PUFA that can be used to cover the deficit in these health claimable fatty acids in Western diets. [ABSTRACT FROM AUTHOR]