Background: We aimed to assess whether whole blood expression quantitative trait loci (eQTLs) with effects in cis and trans are robust and can be used to identify regulatory pathways affecting disease susceptibility. Materials and Methods: We performed whole-genome eQTL analyses in 890 participants of the KORA F4 study and in two independent replication samples (SHIP-TREND, N = 976 and EGCUT, N = 842) using linear regression models and Bonferroni correction. Results: In the KORA F4 study, 4, 116 cis-eQTLs (defined as SNP-probe pairs where the SNP is located within a 500 kb window around the transcription unit) and 94 trans-eQTLs reached genome-wide significance and overall 91% (92% of cis-,84% of trans-eQTLs) were confirmed in at least one of the two replication studies. Different study designs including distinct laboratory reagents (PAXgene (TM) vs. Tempus (TM) tubes) did not affect reproducibility (separate overall replication overlap: 78% and 82%). Immune response pathways were enriched in cis- and trans-eQTLs and significant cis-eQTLs were partly coexistent in other tissues (cross-tissue similarity 40-70%). Furthermore, four chromosomal regions displayed simultaneous impact on multiple gene expression levels in trans, and 746 eQTL-SNPs have been previously reported to have clinical relevance. We demonstrated cross-associations between eQTL-SNPs, gene expression levels in trans, and clinical phenotypes as well as a link between eQTLs and human metabolic traits via modification of gene regulation in cis. Conclusions: Our data suggest that whole blood is a robust tissue for eQTL analysis and may be used both for biomarker studies and to enhance our understanding of molecular mechanisms underlying gene-disease associations.