Cryptococcosis is a systemic infection caused by the pathogenic yeasts Cryptococcus neoformans and C. gattii. Detection of cryptococcal capsular antigen (CrAg) in serum and cerebrospinal fluid (CSF) plays an important diagnostic role. We prospectively compared the new Immuno-Mycologics Inc. (IMMY) lateral flow assay (LFA) and enzyme immunoassay (EIA) to our current CrAg test (Premier EIA; Meridian Bioscience Inc.). Discordant samples were retested with the latex-Cryptococcus antigen test (IMMY) and using serotype-specific monoclonal antibodies (MAbs). A total of 589 serum and 411 CSF specimens were tested in parallel. Qualitative agreement across assays was 97.7%. In all, 56 (41 serum and 15 CSF) samples were positive and 921 (527 serum and 394 CSF) samples were negative by all three assays. The 23 discrepant specimens were all Meridian EIA negative. Of 23 discordant specimens, 20 (87.0%) were positive by both the IMMY LFA and EIA, 2 were LFA positive only, and 1 was EIA positive only. Eleven discrepant specimens had adequate volume for latex agglutination (LA) testing; 8 were LA positive, and 3 were LA negative. LA-negative samples (2 CSF samples and 1 serum) had low IMMY LFA/EIA titers (≤1:10). Serotype-specific MAb analysis of the LA-positive samples suggested that these specimens contained CrAg epitopes similar to those of serotype C strains. In conclusion, the IMMY assays showed excellent overall concordance with the Meridian EIA. Assay performance differences were related to issues of analytic sensitivity and possible serotype bias. Incomplete access to patient-level data combined with low specimen volumes limited our ability to fully resolve discrepant results.