The Immulite 2000xpi, a chemiluminescent immunoassay (CLIA)-based analyzer, is currently recommended for measuring thyroid hormones (TH) concentrations in equids. However, scarce data are available on TH measurements in donkeys using this technique. Similarly, the effects of factors such as age and gender on TH concentrations have not been assessed with this analyzer. Serum free and total T4 and T3 concentrations were measured in 40 healthy donkeys (6.1 ± 0.8 years old; 8 intact jacks and 32 jennies) and 41 horses (10.7 ± 0.5 years old; 15 stallions and 26 geldings) using the Immulite 2000xpi. A Mann–Whitney test was used to compare two groups, and a Kruskal–Wallis test followed by Dunn’s post hoc test was used for comparisons among more than two groups. This analyzer had lower intra-assay variability and fewer measurements under the detection limit for tT4 and fT4 than for tT3 and fT3 determinations. The canine TSH kit for thyroid-stimulating hormone (TSH) showed high imprecision. Donkeys had higher serum TH concentrations than horses. No differences were observed among gender or age groups. This is the first study to report TH concentrations in donkeys using a CLIA-based analyzer and to evaluate the effects of several factors on TH concentrations. The canine TSH kit is not valid for measuring TSH in equids.