Lactate, the primary byproduct of glycolysis, has been established as a critical barometer of tumor microenvironment homeostasis and is implicated in tumor progression. Alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) have been identified as sensors of intracellular L-lactate and as contributors to cancer development. Nonetheless, investigations into the different roles of AARS1 and AARS2 across various cancer types remain lacking. In this study, we preliminarily explored the correlations between AARS1/2 and the tumor microenvironment in pan-cancer. We first examined the expression patterns of AARS1 and AARS2 across 33 cancer types. Subsequently, we analyzed the relationship of AARS1/2 with clinical features and mutational landscape utilizing the GSCA and cBioPortal databases. Survival outcomes associated with AARS1/2 were assessed through Cox regression and the Kaplan-Meier method. Additionally, we examined the correlations between AARS1/2 and drug sensitivity, as well as immune infiltration, using the GSCA database, TISIDB database, and the CIBERSORT algorithm, respectively. Notably, significant heterogeneity of AARS1 and AARS2 was observed across various dimensions, including expression profiles, clinical outcomes, mutation spectra, and immune infiltration. The expression patterns of AARS1/2 were statistically different in urologic neoplasms. Similarly, survival analysis revealed a close correlation between elevated AARS1 expression and unfavorable prognosis in patients with urologic neoplasms, while AARS2 expression level was not. Interestingly, the results of immune infiltration indicated the statistical heterogeneity of AARS1 and AARS2 in certain urologic neoplasms, especially in bladder urothelial carcinoma (BLCA). Specifically, (1) the correlation between AARS1/2 expression and immune-related scores in BLCA was opposite; and (2) TIDE analysis demonstrated that elevated AARS1 expression was related to a high TIDE score in BLCA, while AARS2 was not. Further detailed analysis of AARS1 and AARS2 in the context of immune infiltration and functional enrichment in BLCA may partially account for these observations. In conclusion, our findings highlighted the heterogeneity of AARS1 and AARS2 across various cancers. Based on our data, AARS1 was identified as a potential prognostic biomarker and a candidate for immunotherapy targeting in urologic cancers, though further validation is needed to confirm its clinical utility.© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.