The four chambers of the human heart play distinct roles in the maintenance of normal cardiac function, and are differentially affected by inherited/acquired cardiovascular disease. To probe the molecular determinants of these functional differences, we examined mRNA and lncRNA expression profiles in the left (LA) and right (RA) atria, the left (LV) and right (RV) ventricles, and the interventricular septum (IVS) of non-failing human hearts (N = 8). Analysis of paired atrial and ventricular samples (n = 40) identified 5,747 mRNAs and 2,794 lncRNAs that were differentially (>1.5 fold; FDR < 0.05) expressed. The largest differences were observed in comparisons between the atrial (RA/LA) and ventricular (RV/LV/IVS) samples. In every case (e.g., LA vs LV, LA vs RV, etc.), >2,300 mRNAs and >1,200 lncRNAs, corresponding to 17–28% of the total transcripts, were differentially expressed. Heterogeneities in mRNA/lncRNA expression profiles in the LA and RA, as well as in the LV, RV and IVS, were also revealed, although the numbers of differentially expressed transcripts were substantially smaller. Gender differences in mRNA and lncRNA expression profiles were also evident in non-failing human atria and ventricles. Gene ontology classification of differentially expressed gene sets revealed chamber-specific enrichment of numerous signaling pathways.