Retinal neurotransmitters are known to play a role in postnatal ocular development and eye growth. The success of muscarinic antagonists in blocking form-deprivation myopia has implicated retinal acetylcholine in the control of ocular growth. The present study investigated whether steady-state content of acetylcholine (ACh) and its metabolite choline (Ch) are altered in the retina of eyes developing axial myopia, in both tree shrews and chicks. Retinal ACh and Ch content were measured using reverse-phase high-performance liquid chromatography. Posterior and anterior retinal samples were analyzed from myopic (form deprived) and control eyes as well as age-matched normal eyes. Normative data on retinal neurotransmitter content demonstrated that chick retinas contained less than half the ACh and Ch neurotransmitter content of tree shrews when normalized to retinal protein (ACh: 61 ± 3 vs. 130 ± 6 ng, Ch: 131 ± 5 vs. 347 ± 25 ng). There was no significant difference in either ACh or Ch content between myopic and contralateral control eyes in either tree shrews or chicks, irrespective of the degree of myopia. This finding was consistent for both posterior, anterior, and consequently whole retinal samples. In contrast, dopamine and DOPAC contents were found to be reduced in myopic compared to control eyes of the same tree shrews (dopamine −6.9% and DOPAC −15.5%) and chicks (dopamine −12.3% and DOPAC −28.2%). These findings demonstrate that, contrary to dopamine and DOPAC content, steady-state retinal acetylcholine and choline content is not significantly altered during myopia development.