During embryonic development of the visual system, retinal ganglion cells (RGCs) project their axons towards the brain, passing through the optic chiasm. Axons are guided on this journey by molecular cues in the environment. The heparan sulphate sulphotransferase (Hst) enzymes Hs2st and Hs6st1 are each known to be required for specific aspects of axon guidance in the developing visual system, as revealed by studies of Hs2st(-/-) and Hs6st1(-/-) mutant embryos. However, it remained possible that these two enzymes have additional, overlapping, functions in RGC axon guidance; but that no effect is manifest in single mutant embryos, because the other enzyme is sufficient to fulfil the shared function. To investigate this possibility, we generated a set of Hs2st; Hs6st1 double mutant embryos that had reduced gene dosage of each of these Hsts, reasoning that any additional phenotypes in these animals would indicate the presence of functional overlap. We first characterised the structure of the mutant Hs6st1 locus, identifying the insertion site of the gene trap vector, to allow us to genotype compound mutants reliably. We found that Hs2st(-/-); Hs6st1(-/-) mutants that lack both enzymes died prior to E15.5. As the optic chiasm has not formed by this stage, we were unable to determine the effect of complete loss of Hs2st and Hs6st1 on chiasm formation. However, compound mutant embryos lacking one Hst and heterozygous for the other were viable. We found that RGC axon guidance defects in such compound mutants were no more severe than those found in the single mutant embryos. We also found that expression of the Hs6st1 isoform Hs6st3 overlaps with that of Hs6st1 in the developing visual system, suggesting that some Hs6st activity remains present in this region of Hs6st1(-/-) mutant embryos.