The high concentration of ammonia from deteriorated aquaculture environments and the intensive culture system could increase the susceptibility to pathogens and even cause high mortality in Litopenaeus vannamei. In addition, we have revealed that the ammonia-tolerant shrimp also have high disease resistance in L. vannamei. In the present study, in order to identify SNP markers associated with tolerance to ammonia toxicity, we developed and characterized SNPs from our previous transcriptome sequencing data of ammonia-stressed and control groups, and a marker-trait association analysis was performed for marker-assisted selection (MAS) to increase production in L. vannamei. A total of 318,919 SNPs were identified from the transcriptome sequences, and 25,772 SNPs were found from the 1826 ammonia-responsive genes with functional annotation. We selected 49 SNPs from 26 ammonia-responsive genes that had strong homologies to known genes in the shrimp and probably involved in immune function as candidate markers for genotyping, among which 39 SNPs were polymorphic for further marker-trait association analysis with the ammonia-tolerant (AT) and ammonia-sensitive (AS) groups. Finally, 12 out of the 49 SNP markers were identified to be associated with ammonia tolerance, containing 10 loci with significantly different allele frequencies and 10 loci with significantly different genotyping frequencies between the AT and AS groups. Among the associated markers, the G allele of TSP-1 (the first locus from the thrombospondin gene), the A allele of TSP-3, and the C allele of XBP1–5 (the fifth locus from X-box binding protein 1) only presented in the AT groups, but they were absent from the AS groups, which would be the preference of the MAS for the ammonia-tolerant shrimp. In addition, when the 12 associated SNP markers were used for analysis, the genetic diversity of the AT groups were significantly higher than that of the AS groups, but when the 39 loci were used there was no difference. This is the first report for the markers associated with ammonia tolerance in this species, indirectly with disease resistance, which provided important potential for genetic selection to increase survival rate and production in shrimp farming.