Whole genome association studies in humans have shown a strong relationship between omega-3 levels in plasma and single nucleotide polymorphisms (SNP) located close to genes whose protein products are involved in the biosynthesis of long-chain omega-3 fatty acids. In sheep and other livestock species, the calpain/calpastatin system is the principal influence on natural variation in meat tenderness between animals. Using targeted next generation sequencing, we sequenced the fatty acid desaturase locus (FADS1/2/3), ELOVL2 and SLC26A10 and the calpain/calpastatin (CAPN1/2/3 and CAST) gene loci of 35 industry sires from the Australian flock. A total of 753 SNP were identified and 182 of these SNP were selected for incorporation onto a research SNP panel that represented the genetic variation across the nine genes. A total of 1252 animals were genotyped from the Australian Sheep CRC Information Nucleus Flock for these SNP and the genomic association was calculated for omega-3 fatty acid content and objective meat tenderness in lamb. Six SNP within CAST and CAPN2 showed association with shear force at Day 5 post-mortem at a significance level of P ≤ 0.01. When these were fitted simultaneously in a mixed-model analysis with fixed effects and covariates, three SNP remained significant. These SNP each had an unfavourable effect on shear force of between 1.1 and 1.8 N, with a combined effect of 4.1 N. The frequency of the favourable alleles in the progeny measured indicates that these SNP hold good potential for improving the management of meat tenderness across Merino, Border Leicester and Terminal sire types. No SNP within the FADS1/2/3, ELOVL2 and SLC26A10 gene regions were associated with lamb muscle omega-3 levels. This indicates that genetic variation in the long-chain omega-3 biosynthesis pathway genes analysed here may not be important for omega-3 content in lamb within the Information Nucleus Flock population.